Three dimensional matrix for cancer stem cells

ABSTRACT

Synthetic inert 3D gel culture systems are described that can be finely tuned to exhibit desired and predetermined physical, chemical, mechanical, and biochemical properties. The culture system can be utilized to study the effect of microenvironmental factors on cancer cell response, and in particular on cancer stem cell (CSC) response. Cancer cells can be encapsulated in a crosslinked gel system having a narrow range of predetermined gel stiffness. One or more biochemical factors including peptides that can affect the growth, development, and/or proliferation of CSCs can be incorporated in the system to examine the effects of the factor(s) on the encapsulated cells with regard to growth, proliferation, size, etc.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims filing benefit of U.S. Provisional PatentApplication Ser. No. 61,962,057 entitled “Engineered matrix forEnriching Malignant Cancer Stem Cells” having a filing date of Oct. 30,2013 and U.S. Provisional Patent Application Ser. No. 61/962,056entitled “Regulating Cancer Stem Cell Maintenance with Integrin andHeparin Binding Peptides” having a filing date of Oct. 30, 2013, both ofwhich are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under CBET-0931998,CBET-0756394 and DMR-1049381 awarded by the National Science Foundationand under 1R03DE019180-01A1 awarded by the national Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 12, 2014, isnamed USC-457(1067)_SL.txt and is 7,517 bytes in size.

BACKGROUND

Breast cancer is the most common cancer among women in industrializedcountries. The development of breast cancer is a multiple-step processand regulated by the tumor microenvironment. This development processmay take many years and is difficult to follow in vivo, Therefore, thereis a need to develop in vitro models to study the molecular basis oftumorigenesis and progression in breast cancer as well as in othercancers.

Most in vitro cancer cell studies use standard two-dimensional (2D) cellculture systems. However, cells grown on 2D tissue culture behavedifferently from those grown in a physiological three-dimensional (3D)environment due to the lack of proper cell-cell and cell-matrixinteractions as well as the lack of gradient of nutrients and growthfactors, which are known to play critical roles in cancer initiation,progression and metastasis. For example, when cancer cells are culturedin 2D plates, their malignancy is reduced compared to those under invivo conditions. Animal models are also frequently used to studymolecular pathways and drug response in cancer research. In these cases,either animal tumors grown in syngeneic animals or human tumors grown inimmunocompromised animals are used. Therefore, animal models may notadequately reproduce the features of human cancers in vivo.

To bridge the gap between the 2D cell culture system and the in vivosystem, the 3D in vitro cell culture system has emerged. In many 30models, cell lines or cells from dissociated tissues are embedded in 3Dmatrices and cultured to promote cell-cell interaction, adhesion,migration and in vivo-like morphogenesis. Comparison between 2D and 3Dculture systems has revealed significant differences in all aspects ofcell behavior from cell shape and growth to gene expression and responseto stimuli. Various types of materials have been used to generate a 3Dmatrix. Type I collagen and Matrigel™ are the most widely used matricesbecause they are biocompatible and support adhesion and growth of manycell types. Alginate and agarose gels are also used as a matrix to studythe behavior of cancer cells under 3D conditions. Unfortunately, it isdifficult to control the physical characteristics of gels formed ofnaturally derived polymers. In addition, it is difficult to isolate andstudy cell response to individual factors in these microenvironments asthe naturally derived matrices tend to interact with surface receptorsof the cells.

As a result, inert synthetic polymers have been examined for use indevelopment of 3D gels. The use of inert synthetic polymers can provideincreased flexibility in designing 3D matrices with a wide range ofmechanical, physical, and biological properties. Among the syntheticmaterials, polyethylene glycol (PEG) hydrogel, due to its inert nature,has been used extensively to form engineered matrices for cellencapsulation. While the development of PEG 3D matrices has been animprovement in the art, room for further improvement exists.

For example, while the effect of matrix stiffness on the response ofnormal stem cells has been studied, the effect of matrix stiffness oncancer stem cells (CSCs) encapsulated within an inert microenvironmenthas not been investigated. Normal stem cells and cancer stem cells usesimilar signaling pathways to maintain their sternness, However, theymay respond to the environmental cues differently. The microenvironmentor niche under normal conditions inhibits stem cell proliferation anddifferentiation, but cancer stem cells, due to mutations in the cell,are self-sufficient with respect to proliferation, It has been proposedthat the stem cell niche is converted from proliferation inhibitory toone favoring cell proliferation in the case of cancer stem cells. Whatis needed in the art is a tunable 3D matrix that can be utilized toexamine such propositions for further understanding the growth anddevelopment of cancer cells, and in particular cancer stem cells, e.g.,a 3D matrix that can be utilized to enrich a cell sample in cancer stemcells. For instance, the fraction of CSCs in the population of cancercells is understood to be at most a few percent, and possibly less than1%. As a result, drug toxicity tests to date evaluate the response ofnon-stem-like cancer cells to the chemotherapy agent. Unfortunately,CSC's are the cell fraction responsible for cancer recurrence, relapse,and metastasis and the CSC fraction is the most malignant fraction ofcells in the population of cancer cells. Therefore, there is a need todevelop technologies and 3D matrices that can be utilized to enrich acell population in cancer stem cells for study and drug testing.

In addition to the need to develop improved 3D matrices, as cancer cellsare affected by many factors in their microenvironment, another majorchallenge to understanding the growth and development of cancer cellslies in developing methods to isolate the effect of single factors onparticular cell types while keeping other factors unchanged. Forinstance, breast tumors are highly heterogeneous, and cells withself-renewal and highly invasive capacity coexist with cells that aremore differentiated and non-invasive. Increasing evidence suggests thatthe heterogeneity of the tumor tissue is rooted in the existence ofCSCs. Therefore, understanding the mechanism of CSC maintenance, and inparticular the effect of specific factors on CSC maintenance andenrichment, is critical for breast cancer prevention and treatment.

Cell to cell interactions between stem cells and support cells,interactions between stem cells and extracellular matrix (ECM), thecomposition of ECM and the physicochemical properties of the environmentare all key contributing factors in stem cell maintenance. Many in vitrostudies have provided insight on the regulation of CSC fate by themicroenvironment. However, these studies have been limited by the natureof the support matrix as well as by the inability to isolate the effectof single factors in a realistic model. Accordingly, what is also neededin the art is a 3D matrix having a highly controlled microenvironment soas to more accurately isolate and determine the effects of particularfactors on the growth and development of cancer cells, and inparticular, of stem cancer cells.

SUMMARY

According to one embodiment, disclosed is a method of forming a threedimensional hydrogel matrix for supporting a cancer stem cell. Forinstance, the method can include combining an inert synthetic polymerwith a crosslinking agent to form a precursor solution and crosslinkingthe inert synthetic polymer via the crosslinking agent to form the threedimensional hydrogel matrix. More specifically, the concentration of thecrosslinking agent and/or the concentration of the inert syntheticpolymer can be predetermined in the precursor solution such that thethree dimensional hydrogel matrix has a predetermined elastic modulus.In addition, a method can include conjugating a peptide to the matrixthat can effect the growth, development, and/or proliferation of acancer stem cell. For instance, an integrin binding peptide and/or aCD44 binding peptide can be conjugated to the matrix, either of whichcan be utilized to prevent the proliferation of CSC's (i.e., turn offthe CSC's) encapsulated in the matrix. Alternatively, a heparin bindingpeptide can be conjugated to the matrix, which can be utilized topromote the proliferation of CSC's (i.e., “turn on” the CSC's) in thematrix.

Also disclosued is a three dimensional hydrogel matrix comprising acrosslinked inert synthetic polymer. The hydrogel matrix can alsoinclude a peptide conjugated to the matrix, the peptide being one thatcan affect the growth, development, and/or proliferation of a CSCencapsulated in the matrix. The three dimensional hydrogel matrix has anelastic modulus that is predetermined, for instance less than about 10kilopascals in one particular embodiment.

Also disclosed are methods of utilizing the three dimensional hydrogelmatrices for study of a cell population, for instance a cell populationthat includes cancer cells and cancer stem cells, optionally inconjunction with other cell types. For example, in one embodiment thethree dimensional hydrogel matrix can include an integrin bindingpeptide (e.g., GRGDS, SEQ ID NO.: 19) or a mutant thereof, or the threedimensional hydrogel matrix can include a CD44 binding peptide (e.g.,RLVSYNGIIFFLK, SEQ ID NO.: SEQ ID NO.: 17) or a mutant thereof, and thematrix can be utilized to turn off CSC's in the cell populationencapsulated in the matrix. In another embodiment, the hydrogel matrixcan include a heparin binding peptide (e.g., WQPPRARI (SEQ ID NO,: 21)or a mutant thereof, and the matrix can be utilized to turn on CSC's ina cell population that is encapsulated in the matrix.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a reaction scheme for acrylation of aPEG macromer (FIG. 1A) and the ¹H-NMR spectrum of PEGDA macromer (FIG.1B). The chemical shifts between 5.85 and 6.55 ppm due to acrylatehydrogens are enlarged in the inset of the NMR spectrum.

FIG. 2 graphically illustrates the effect of macromer concentration onelastic modulus of 4T1 cell loaded (1.4×10⁵ cells/mL) PEGDA hydrogelswith incubation time. Error bars correspond to means±1 SD for n=3.

FIG. 3 compares in vivo tumor formation of 4T1 cells from adhesionplates (FIG. 3A) with 4T1 cells from tumorspheres on ultra-lowattachment plates (FIG. 3B). The left and right images in FIG. 3A showtumor formation by inoculation of 5000 and 50,000 4T1-Luc cells,respectively, The left, center, and right images in FIG. 3B show tumorformation by inoculation of 500, 1000, and 5000 4T1-luc cells fromtumorspheres, respectively. 4T1-Luc cells were inoculated subcutaneouslyin Balblc mice. After 1 week, the expression of luciferase in tumors wasimaged.

FIG. 4 presents live and dead images of 4T1 cells 2 days afterencapsulation in PEGDA hydrogels with moduli of 2.5 kPa (FIG. 4A), 5.3kPa (FIG. 4B), 26.1 kPa (FIG.b 4C) and 47.5 kPa (FIG. 4D). The scalebars in images of FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D are 200 μm.Based on image analysis, the percent viable cells for the 2.5, 5.3,26.1, 47.5 and 68.6 kPa gels was 94±4, 91±3, 92±3, 90±4, and 89±4,respectively. FIG. 4E1, FIG. 4E2, FIG. 4E3, FIG. 4E4, FIG. 4E5, FIG.4E6, FIG. 4E7, and FIG. 4E8 on the right show the uniformity of cellseeding and cell viability in successive 90 μm layers in the directionof thickness for 5.3 kPa gel, obtained with a confocal fluorescentmicroscope.

FIG. 5 illustrates the evolution of tumorsphere formation by 4T1 tumorcells encapsulated in PEGDA hydrogels with elastic modulus of 2.5 kPa(left column), 5.3 kPa (center left column), 47.5 kPa (center rightcolumn), and MCF7 tumor cells encapsulated in the 5.3 kPa gel (leftcolumn) as a function of incubation time. Rows 1, 2, 3, and 4 correspondto incubation times of 5, 8, 11, and 14 days, respectively. At each timepoint, encapsulated cells were stained with phalloidin for cytoskeletonand DAPI for nucleus, and imaged with an inverted fluorescentmicroscope.

FIG. 6 presents representative images of tumorsphere density for 4T1(left column) and MCF7 (right column) cells encapsulated in PEGDAhydrogels with elastic modulus of 2.5 kPa (first row), 5.3 kPa (secondrow), 26.1 kPa (third row), and 47.5 kPa (fourth row) after 8 days ofincubation. At each time point, encapsulated cells were stained withphalloidin for cytoskeleton and DAPI for nucleus, and imaged with aninverted fluorescent microscope.

FIG. 7 presents average tumorsphere size (FIG. 7A and FIG. 7D),tumorsphere size distribution (FIG. 78 and FIG. 7E), and cell count(FIG. 7C and FIG. 7F) for 4T1 and MCF7 tumor cells encapsulated in PEGDAhydrogels with different elastic moduli and incubated for up to 14 days.Graphs FIG. 7A, FIG. 78, and FIG. 7C correspond to 4T1 cells and FIG.7D, FIG. 7E, and FIG. 7F correspond to MCF7 cells. The star indicatesstatistically significant difference between the test group and allother groups at the same time point (FIG. 7A, FIG. 7C, FIG. 7D, and FIG.7F) or at the same tumorsphere diameter range (FIG. 7B and FIG. 7E).Error bars correspond to means±1 SD for n=3.

FIG. 8 illustrates BrdU staining of 4T1 tumorspheres formed insuspension culture on low adhesion plates (FIG. 8A and FIG. 8C) andformed by encapsulation in PEGDA hydrogels (FIG. 8B and FIG. 8D). FIG.8A and FIG. 88 were taken after 8 days of incubation while images FIG.8C and FIG. 8D were following 14 days, Prior to tumorsphere formation,4T1 cells were incubated with BrdU for 10 days to achieve stablelabeling. The presence of BrdU in the cells was confirmed byimmunofluorescent staining. The cell nuclei were stained with DAPI.

FIG. 9 presents the effect of gel elastic modulus on the relative mRNAexpression levels of CD44 (FIG. 9A), CD24 (FIG. 9B), ABCG2 (FIG. 9C),Seal (FIG. 9D) markers of 4T1 cells, and CD44 (FIG. 9E), ABCG2 (FIG. 9F)markers of MCF7 cells encapsulated in PEGDA hydrogels with incubationtime. The mRNA expression levels of the markers for 4T1/MCF7 cellsbefore encapsulation were used as reference (set equal to one). The starindicates statistically significant difference between the test groupand all other groups at the same time point. Error bars correspond tomeans±1 SD for n=3.

FIG. 10 illustrates the expression pattern of CD44 marker of 4T1tumorspheres formed in suspension culture on low adhesion plates (columntitled FIG. 10A) and formed by encapsulation in PEGDA hydrogel with 5.3kPa elastic modulus (column titled FIG. 10B), Images in rows 1, 2, 3,and 4 are after 5, 8, 11, and 14 days of incubation, respectively. Thecell nuclei were stained with DAPI.

FIG. 11 illustrates sphere formation and the effect of cell typeencapsulated in PEGDA gels on the expression of CSC markers.Representative fluorescent images of the tumorsphere size anddistribution for 4T1 (FIG. 11A), MCF7 (FIG. 11 B), and MCF10a (FIG. 11C)cells encapsulated in PEGDA gels (1.4×10⁵ cells/ml), and cultured instem cell culture medium. Encapsulated cells were stained withphalloidin for cytoskeleton and DAPI for nucleus. Also shown is theeffect of cell type on cell number density (FIG. 11D) and tumorspherediameter (FIG. 11E) for tumor cells encapsulated in PEGDA hydrogel andincubated in stern cell culture medium for 6 or 9 days. The sphere sizedistribution (FIG. 11F) was determined 9 days after encapsulation.Effect of cell type on CD44 (FIG. 11G), CD24 (FIG. 11H) and ABCG2 (FIG.11I) mRNA marker expression for tumor cells encapsulated in PEGDAhydrogel and incubated in stem cell culture medium for 6 or 9 days. RNAlevels of the cells were normalized to those at time zero, A starindicates a statistically significant difference (p<0.05) between thetest group and the groups with different cell type in the same timepoint (the same diameter range in f). Values are expressed as mean±SD(n=3).

FIG. 12 illustrates viability of cells encapsulated in PEGDA gel andincludes representative images of live and dead 4T1 cells encapsulatedin PEGDA gels with 5 kPa modulus and cultured in stem cell culturemedium for 2 (FIG. 12A), 6 (FIG. 12B and 12 (FIG. 12C) days. Cells werestained with cAM/EthD for live and dead cell imaging. The insets in FIG.12A, FIG. 12B, and FIG. 12C are live/dead images of 4T1 cells in PEGDAgels with 70 kPa modulus after 2, 6, and 12 days, respectively.

FIG. 13 illustrates CSC population in the cells encapsulated in PEGDAgel. MCF7 cells were encapsulated in PEGDA gels with 5 kPa modulus andcultured in stem cell culture medium. Cells before encapsulation (FIG.13A), 3 days (FIG. 13B), 8 days (FIG. 13C) and 11 days (FIG. 13D) afterencapsulation were stained with CD44-FITC and CD24−PE antibodies. Thepopulation of CD24+, CD44+ and CD44+/CD24− cells was determined by flowcytometry. Flow cytometry was repeated multiple times on each sample toascertain reproducibility of the results.

FIG. 14 illustrates the effect of CD44BP on tumorsphere formation andCSC marker expression. Representative fluorescent images of thetumorsphere size and distribution for 4T1 cells encapsulated in PEGDAgels (1.4×10⁵ cells/ml) conjugated with CD44BP (FIG. 14A, conj CD44BP),conjugated with a scrambled sequence of CD44BP (FIG. 14B, conjs-CD44BP), CD44BP dissolved in the gel (FIG. 14C, dis CD44BP), ands-CD44BP dissolved in the gel (FIG. 140, dis s-CD44BP) and cultured inthe stem cell culture medium for 9 days. Effect of CD44BP on cell numberdensity (FIG. 14E) and tumorsphere number density (FIG. 14F) for 4T1tumor cells encapsulated in PEGDA hydrogel and incubated in the sterncell culture medium for 9 days. Effect of CD44BP conjugation on CD44(FIG. 14G), CD24 (FIG. 14H), ABCG2 (FIG. 14I) and SCA1 (FIG. 14J) mRNAmarker expression for 4T1 tumor cells encapsulated in PEGDA gel andincubated in the stem cell culture medium for 9 days. RNA levels of thecells were normalized to those at time zero. A star indicates astatistically significant difference (p<0.05) between the test group and“Ctrl”. Two stars indicates a significant difference (p<0.05) betweenthe two CD44BP and s-CD44BP groups within the same form of peptideaddition (Dis or Conj). Values are expressed as mean±SD (n=3).

FIG. 15 illustrates the effect of CD44BP conjugated to the gel on tumorformation in vivo. The gel without cell (negative control, triangle),4T1 tumorspheres in suspension (positive control, square), 4T1 cellsencapsulated in the gel without CD44BP (circle), and 4T1 cellsencapsulated in the gel with CD44BP (triangle) were inoculatedsubcutaneously in syngeneic Balb/C mice. Tumor sizes were measured dailyfrom post-inoculation day 11 (n32 6/group). Tumor growth was notobserved in the negative control group (the gel without cell) and thegroup with 4T1 cells in the gel with CD44BP (the lines for these twogroups are overlapped in the figure).

FIG. 16 illustrates the comparison of tumorsphere formation in PEGDAgels conjugated with CD44BP, IBP, or FHBP. Representative fluorescentimages of the tumorsphere size and distribution for 4T1 cellsencapsulated in PEGDA gels (1.4×10⁵ cells/ml) without peptideconjugation (FIG. 16A), conjugation with CD44BP (CD44BP, FIG. 16B),conjugation with RGD integrin-binding peptide (IBP, FIG. 16C) andconjugation with fibronectin-derived binding peptide (FHBP, FIG. 16D)and cultured in the stem cell culture medium for 9 days. Effect of cellbinding peptide on cell number density (FIG. 16E), tumorsphere numberdensity (FIG. 16F) and sphere size distribution (FIG. 16G) for 4T1 tumorcells encapsulated in PEGDA gel and incubated in the stem cell culturemedium for 9 days. Effect of cell binding peptide on CD44 (FIG. 16H),CD24 (FIG. 161) and EGFR (FIG. 16J) mRNA marker expression for 4T1 tumorcells encapsulated in PEGDA hydrogel and incubated in the stem cellculture medium for 9 days. RNA levels of the cells were normalized tothose at time zero, A star indicates a statistically significantdifference (p<0.05) between the test group and “Ctrl”. Two starsindicates a significant difference (p<0.05) between the wild type andscrambled peptides for the same conjugated peptide. Values are expressedas mean±SD (n=3).

FIG. 17 illustrates CSC population in cells encapsulated in PEGDA gelsconjugated with CD44BP, IBP, or FHBP. 4T1 cells were encapsulated inPEGDA gels with 5 kPa modulus and cultured in stem cell culture medium.Cells before encapsulation (FIG. 17A), 9 days after encapsulation in thegel without peptide (FIG. 17B), 9 days in the gel conjugated with FHBP(FIG. 17C), 9 days in the gel conjugated with !BP (FIG. 17D), and 9 daysin the gel conjugated with CD44BP were stained with CD44-FITC and CD24−PE antibodies (FIG. 17E). The population of CD24+, CD44+ and CD44+/CD24−cells was determined by flow cytometry. Flow cytometry was repeatedmultiple times on each sample to ascertain reproducibility of theresults.

FIG. 18 illustrates expression of the markers related to CSC maintenancein cells grown in the gel conjugated with CD44BP, IBP, or FHBP. Effectof cell binding peptide on N-Cadherin (FIG. 18A), E-Cadherin, (FIG.18B), integrin aV (FIG. 18C), and integrin β3 (FIG. 180) mRNA markerexpression for 4T1 tumor cells encapsulated in the PEGDA hydrogel andincubated in the stem cell culture medium for 9 days. Effect of cellbinding peptide on vimentin (FIG. 18E) and VEGF (FIG. 18F) proteinexpression, The protein expression was determined by western blot andquantified with imageJ. Actin was used as the internal control and theprotein expressions were normalized to those at time zero. RNA levels ofthe cells were normalized to those at time zero. A star indicates astatistically significant difference (p<0.05) between the test group and“Ctrl”. Values are expressed as mean±SD (n=3).

DETAILED DESCRIPTION

The following description and other modifications and variations to thepresently disclosed subject matter may be practiced by those of ordinaryskill in the art, without departing from the spirit and scope of thedisclosure. In addition, it should be understood that aspects of thevarious embodiments may be interchanged in whole or in part.Furthermore, those of ordinary skill in the art will appreciate that thefollowing description is by way of example only, and is not intended tolimit the invention.

The present disclosure is generally directed to a synthetic inert 3D gelculture system that can be finely tuned to exhibit desired andpredetermined physical, chemical, mechanical, and biochemicalproperties. The culture system can be beneficially utilized to study theeffect of microenvironmental factors on cancer cell response and inparticular on cancer stem cell (CSC) response. For instance, CSCs formedin a gel system having a narrow range of predetermined gel stiffnessfollowing encapsulation of breast cancer cells in the gel can maintaintheir sternness over time. This effect can be varied by variation in thestiffness of the gel.

As utilized herein, the term ‘gel stiffness’ and ‘matrix stiffness’ areutilized interchangeably and generally refer to the elastic modulus ofthe gel. Determination of elastic modulus of a gel can be carried outaccording to standard practices, for instance by loading a sample of thegel on the Peltier plate of a rheometer and subjecting the gel touniaxial compressive force. The slope of the linear fit to thestress-strain curve can be taken as the elastic modulus (E) of the gel.

In addition, the disclosed culture systems can be beneficially utilizedto examine the effects of microenvironmental factors on CSCs and on cellpopulations that are enriched in or alternatively depleted in CSCs. Inparticular, the synthetic inert 3D gel culture system can be formed toinclude a peptide that can affect the proliferation of CSCs in a cellpopulation encapsulated in the system. As such, the system can beutilized to better understand the effects of microenvironmental factorssuch as potential cancer treatment methods and materials on a cellpopulation and in particular on a cancer cell population.

Among the microenvironmental factors of both a 2D and 3D culture system,matrix stiffness is generally known to play an important role inregulating cell function. In vivo, cells have the ability to sense andrespond to matrix stiffness by synthesizing the appropriate ECMcomposition. As the mechanical properties and composition of hard andsoft tissues differ significantly, cells need to respond appropriatelyto environmental cues such as matrix stiffness for survival, Likewise,the proliferation, differentiation, migration, and apoptosis ofcancerous cells in the tumor tissue are regulated by microenvironmentalfactors including matrix stiffness,

In the disclosed 3D culture systems, control of the elastic modulus ofthe matrix can be utilized to better understand and directdifferentiation of encapsulated cells as well as to shift the balance ofcell proliferation and apoptosis. This ability combined with the abilityto include in the inert gel matrix only particularly selectedbiochemical factors can provide a culture system that can be used togreatly enhance the growth of CSCs or to greatly reduce the growth ofCSCs. Moreover, the gels can be formed of biocompatible materials and assuch can be utilized both in vivo and in vitro.

The disclosed matrices can be utilized in one embodiment to investigatethe effect of matrix elastic modulus on the formation, growth, andmaintenance of cancer cells (e.g., CSCs) by encapsulation of tumor cellsin the hydrogel matrix in the absence of attached ligands that caninteract with cell surface receptors. For example, and as furtherdescribed herein, the disclosed matrices have been utilized toillustrate that the formation and maintenance of 4T1 mouse breast cancercells and MCF7 human breast cancer cells can be modulated merely by theelastic modulus of the matrix.

In addition, the disclosed matrices can be utilized to selectivelyenrich or deplete a cell population in CSCs so as to better understandthe development of tumors in the cell population, to examine the effectsof potential treatment protocols on the cell population, andparticularly on CSCs of a cell population, and so forth. For instance,the presence or absence within the gel of binding proteins or bindingpeptide fragments of entire proteins known to effect CSC growth andproliferation can be utilized to control the relative proportion of CSCsin an encapsulated cell population. This control can then be leveragedto better understand the effect of microenvironmental factors on theencapsulated cell population,

A cell population that can be examined can include a single cell type ormultiple different cell types combined together, as desired. Forinstance, in one embodiment cancer stem cells can be encapsulated on thegel with no other cell types. In another embodiment, a cell populationincluding cancer cells (e.g., breast cancer cells, lung cancer cells,etc.) and cancer stem cells can be encapsulated in a gel. For example,ex vivo tumor tissue including cancer cells and cancer stem cells in aproportion as found in vivo can be supported by the matrix. Of course,non-cancerous cells can also be encapsulated in or supported on a gel,for instance in conjunction with cancer cells and cancer stem cells.Other cells can include, without limitation, support cells or tumorstroma that can function as support for the growth of cancer cells.Support cells can include, without limitation, mesenchymal cells,endothelial cells, immune system cells, lymphatic cells, etc.

Through utilization of the disclosed gels it has been determined thatthe presence of a CD44 binding protein (e.g., RLVSYNGIIFFLK, SEQ ID NO.:17) or a mutant thereof (e.g., VLFGFLKIYSRIN, SEQ ID NO.: 18) conjugatedin the gel can inhibit breast tumorsphere formation in vitro and invivo. In addition, tumorsphere formation in vitro can be enhanced by thepresence of a fibronectin-derived heparin binding protein (FHBP) (e.g.,WQPPRARI, SEQ ID NO.: 21) or a mutant thereof (e.g., RPQIPWAR, SEQ IDNO.: 22) while it can be abolished by the presence of an integrinbinding ROD peptide (IBP), (e.g., GROOS, SEQ ID NO.: 19) or a mutantthereof (e.g., GRDGS, SEQ ID NO.; 20).

The disclosed PEGDA hydrogel culture system can provide a useful tool toinvestigate the individual effect of factors such as these bindingfactors, optionally in conjunction with other biochemical factors in themicroenvironment on cancer cell maintenance, and specifically on CSCmaintenance, without interference of other factors. In turn, this canlead to the development of a 3D culture system with enriched populationof CSCs for, e.g., drug testing.

As mentioned, the disclosed system can be developed with a predeterminedelastic modulus in a well-defined narrow range. This can be very usefulas the gel modulus can have a strong effect on tumorsphere formation andthe effect can be bimodal. For instance, the stiffness of normal humanbreast tissue is lower than 4 kPa while that of cancerous breast tissuecan be up to 40 kPa, and the disclosed matrices can be formed with anelastic modulus so as to reproduce the microenvironment of a biologicalsystem anywhere within this range. For example, the hydrogel matrix canbe designed to have an intermediate elastic modulus of from 10kilopascals (kPa) to 30 kPa, a low modulus of less than 10 kPa, forinstance from 2.5 kPa to 7.5 kPa, or a high modulus of from 10 kPa to 70kPa. In one embodiment, the hydrogel matrix can have a low modulus, butone that is higher than the modulus of normal tissue, which is generallyless than 4 kPa. Thus, the hydrogel matrix can have a modulus of from 4kPa to 6 kPa or a modulus of about 5.3 kPa in one embodiment. Asdescribed further herein, mouse 4T1 and human MCF7 cells encapsulated ina gel with 5.3 kPa modulus formed large tumorspheres at a high densityof tumorspheres, and had high expression of breast CSC markers CD44 andABCG2.

The stiffness of the gel matrix can be adjusted so as to replicate theECM stiffness of the encapsulated cells when in vivo. The ECM stiffnessis known to regulate proliferation and differentiation of many celltypes. For instance, development of solid tumors is often accompanied byan increase in the stiffness of the local environment, and a high tissuedensity is a known risk factor for developing invasive breast carcinoma.Analysis of several cell lines in collagen matrices has revealed thatmatrix stiffness can dramatically affect the growth of certain celllines but has little effect on others. For example, the growth ofMDA-MB-231 cells, a highly malignant human breast cancer cell line, issignificantly enhanced with increasing matrix stiffness while the growthof MCF-10a, a nonmalignant breast epithelial cell line, is relativelyinsensitive to matrix stiffness within a certain range, These resultssuggest that the response of cancer cells to matrix stiffness is notonly dependent on the cancer but also on malignancy of the cancer.Furthermore, it has been reported that non-tumorigenic breast epithelialcells loose cell polarity and increase proliferation when cultured inmatrices with 4.5 kPa stiffness. In addition, normal murine mammarygland or well-differentiated mammary epithelial cells cultured inhigh-density collagen matrices display an invasive phenotype.Mechanistic studies suggest that these mechanically inducedtransformations are associated with enhanced focal adhesion kinase (FAK)followed by FAK-dependent ERK and Rho activity. These pathways have beensuggested as the circuit linking matrix stiffness to cytoskeleton.

The 3D system includes an inert synthetic polymer hydrogel that, in oneembodiment, does not have any cell interaction ligands, thus providing aunique tool to study tumor microenvironment in vitro, as cell adhesionligands are believed to mediate mechanosensing. Without wishing to bebound to any particular theory, the cell response to stiffness in theinert matrix is believed to be through the ECM secreted by theencapsulated cells. For example, human mesenchymal stem cells (hMSC)encapsulated in PEG hydrogels, in the absence of adhesive ligands, cansecret their own ECM through which differentiation is directed bymechanotransduction. The disclosed matrices can be used to furtherelucidate such cellular activities. For instance, breast cancer CSCs areshown in the example section below to maintain their sternness andproliferate while the growth of non-CSCs was inhibited when encapsulatedin the gel within a certain range of elastic moduli. Through variationand control of the elastic moduli of the gels in a predeterminedfashion, further information regarding the interaction of encapsulatedcells with the surrounding microenvironment can be elucidated.

In one embodiment, the synthetic polymer of the hydrogel can be PEG.This is not a requirement of the gel systems, however, and othersynthetic inert and biocompatible polymers can be utilized inconjunction with or alternative to a PEG-based system, For instance, theinert synthetic gel can incorporate other polymers such aspolyhydroxyethyl methacrylate (PHEMA), polyvinylpolypyrrolidone (PVP),and polyvinyl alcohol (PVA). The polymer can be any suitable molecularweight, with the preferred molecular weight depending upon thereactivity of the polymer as well as the targeted elastic modulus of thecrosslinked hydrogel matrix. For instance, the polymer can be a lowmolecular weight polymer having a number average molecular weight ofabout 1,000 Da or less, a midrange molecular weight having a numberaverage molecular weight of from about 1,000 Da to about 10,000 Da, or ahigh molecular weight, having a molecular weight of about 10,000 Da orgreater. For instance, the polymer can be a difunctional polymer havinga molecular weight of about 10,000 Da or less in one embodiment, or fromabout 1,000 Da to about 5,000 Da in some embodiments.

The stiffness of the system can be controlled through the crosslinkdensity and/or the density of polymer chains of the hydrogel network.According to rubber elasticity theory, the gel elastic modulus isproportional to the density of elastically active chains and/or thecrosslink density. Accordingly, the network crosslink density can beincreased in one embodiment by increasing the concentration of thecrosslinking agent in a precursor solution that includes a PEG macromerand the crosslinking agent, leading to the increase in matrix modulus.Likewise, by decreasing the concentration of the crosslinking agent inthe precursor solution, the crosslink density and hence the elasticmodulus of the formed gel can be decreased. Alternatively, the crosslinkdensity and hence elastic modulus of the formed system can be controlledthrough variation in the molecular weight of the polymer included in aformation solution, with a higher molecular weight polymer leading to alower elastic modulus and vice versa for a higher elastic modulus gel.Of course, polymer molecular weight can affect crosslink density of thehydrogel primarily in those embodiments in which the polymer includes alimited number of crosslinking sites, for instance in which the polymeris difunctional with crosslinking sites only at the termini of thepolymer backbone chain.

The particular concentrations of the polymer and/or the crosslinkingagent can vary to obtain a pre-determined elastic modulus, generallydepending upon the reactive characteristics of the polymer. For example,in one embodiment, a precursor solution of a difunctional polymer andcrosslinking agent (either prior to or following reaction of the polymerwith the crosslinking agent) can have a polymer concentration of about10% by weight of the solution or less to form a low elastic modulushydrogel matrix (about 10 kPa or less), a polymer concentration of fromabout 10% by weight of the solution to about 20% by weight of thesolution to form an intermediate elastic modulus hydrogel matrix (about10 kPa to about 30 kPa), and a polymer concentration of about 20% byweight of the solution or greater to form a high elastic modulushydrogel matrix (about 30 kPa or greater).

The crosslinking scheme utilized to form the hydrogel can vary dependingupon the particular polymer used in the system. For instance, acrosslinking agent can be reacted with the polymer prior to gelformation or during gel formation, as desired. For example, in oneembodiment a polymer (e.g., a PEG polymer) can first be functionalizedto form a functional macromer (e.g., a PEG diacrylate) and thefunctional macromer can then be crosslinked to form the gel, forinstance by use of an initiator, e.g., a photoinitiator, and subjectionto suitable energy (e.g., a UV cure).

Alternatively, a crosslinking agent can be combined with a polymer thatincludes reactive functional groups at the time of gel formation, andthe crosslinking agent can form links between and among the polymers toform the hydrogel network. The crosslinking agent can be apolyfunctional compound that can react with functionality of the polymerto form crosslinks within the hydrogel network. In general, thecrosslinking agent can be a biocompatible non-polymeric compound, i.e.,a molecular compound that includes two or more reactively functionalterminal moieties linked by a bond or a non-polymeric (non-repeating)linking component. By way of example, the crosslinking agent can includebut is not limited to diacrylates, di-epoxides, poly-functionalepoxides, diisocyanates, polyisocyanates, polyhydric alcohols,water-soluble carbodiimides, diamines, diaminoalkanes, polyfunctionalcarboxylic acids, diacid halides, halo acrylate monomers, and so forth.For instance, when considering a PEG-based polymer, a non-polymericacryloyl halide can be utilized as a crosslinking agent.

In some embodiments, an initiator is utilized to initiate crosslinkingof the polymer. Initiators can include photo-initiators,thermal-initiators, or chemical initiators. For example, in oneparticular embodiment, a UV-initiator can be utilized. Chemicalinitiators can also be used, such as redox, peroxide, etc. In otherembodiments, other radiation initiation processes, such as gamma rays,e-beam, X-ray, etc., can be utilized, which may not require the presenceof an initiator.

For example, a non-limiting list of UV-initiators which may be usedinclude IRGACURE® 184 (1-hydroxycyclohexyl phenyl ketone), IRGACURE®2959 (4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-methylpropyl)ketone)), andDAROCURE® 1173 (α-hydroxy-α,α-dimethylacetophenone), all commerciallyavailable from Ciba Specialty Chemicals (Terrytown, N.Y.).

Additional examples of suitable initiators (which may bephoto-initiators or thermally activated initiators) may include benzoylperoxide, azo-bis-isobutyro-nitrile, di t-butyl peroxide, brornylperoxide, curnyl peroxide, lauroyl peroxide, isopropyl percarbonate,methylethyl ketone peroxide, cyclohexane peroxide, tutylhydroperoxide,di-t-amyl peroxide, dicumyl peroxide, t-butyl perbenzoate, benzoin alkylethers (such as benzoin, benzoin isopropyl ether, and benzoin isobutylether), benzophenones (such as benzophenone and methyl-o-benzoylbenzoate), actophenones (such as acetophenone, trichloroacetophenone,2,2-diethoxyacetophenone, p-t-butyltrichloro-acetophenone,2,2-dimethoxy-2-phenyl-acetophenone, and p-dimethylaminoacetophenone),thioxanthones (such as xanthone, thioxanthene, 2-chlorothioxanthone, and2-isopropylthioxanthone), benzyl 2-ethyl anthraquinone, methylbenzoylformate, 2-hydroxy-2-methyl-1-phenylpropane-1-one,2-hydroxy-4′-isopropyl-2-methyl propiophenone, .alpha.-hydroxy ketone,tetramethyl thiuram monosulfide, allyl diazonium salt, and combinationsof camphorquinone and ethyl 4-(N,N-dimethylamino)benzoate.

When present, only one initiator is necessary, however, one or moresecond initiators may be utilized, The one or more second initiators canbe photo or chemical initiators.

The amount of initiator can generally be supplied in standard amounts,for instance in the range of about 0.01 to about 5% by weight of thepolymer solution

The elastic modulus of a matrix can be predetermined to a very narrowrange. For instance, the initial increase in tumorsphere size and cellnumber density with elastic modulus may be attributed to a deviationfrom normal tissue stiffness of only about 0.17 kPa. This smalldeviation can lead to matrix reorganization and change in the number andlifetime of integrin mediated interactions and related pathways. It hasbeen reported that the sphere size of breast cancer cells encapsulatedin collagen gels increased with an increase in elastic modulus from 0.17to 1.20 kPa. In another study, it was shown that the proliferation andsphere size of hepatocellular carcinoma cells encapsulated inPEG-collagen gels increased with decreasing elastic modulus from 4 kPa(corresponding to the modulus of healthy liver) to 0.7 kPa. Accordingly,in one embodiment, the gel matrix can be formed with an elastic modulusthat differs from the elastic modulus of a native ECM by about 3 kPa orless, for instance 3,3 kPa, or by 1 kPa or less, for instance from 0.17kPa to 1.20 kPa from that of the native ECM.

A decrease in sphere size and cell number of the encapsulated cancercells with increasing elastic modulus of the hydrogel system can beattributed to a decrease in mesh size and increase in retractive forceof the gel network, leading to a negative contribution on the cellproliferation and sphere formation, The average pore size or the meshsize of the hydrogel can also affect diffusion of nutrients and oxygenand tumor cell motility, For example, HepG2 hepatocellular carcinomacells form larger spheres when encapsulated in gels with larger poresizes.

The mesh size of the gels disclosed herein can be, for example, fromabout 25 nanometers (nm) to about 95 nm, for instance from about 35 nmto about 70 nm. The mesh size can be calculated from the modulus andequilibrium swelling ratio of the gels using the Peppas and Barr-Howellequation, as is known in the art. For instance, as the functionalizedPEG macromer concentration of a formation solution increases from 7.5%to 10, 15, 20, and 25%, the mesh size can decrease from 93±4 nm to 67±3,53±3, 34±2, and 25±2 nm, respectively. Without wishing to be bound toany particular theory, the bimodal effect of gel modulus on tumorsphereformation may be due to the changes in the network mesh size that canaffect cell-matrix interactions and nutrient diffusion as well due tochanges in the gel modulus,

Unlike collagen and other biological gels, varying macromerconcentration in the disclosed systems changes only the gel stiffness,not the ligand density in the matrix. Results detailed below of use ofthe disclosed system suggest that the stiffness of the tumor tissuealone can be sufficient to affect the fate of tumor cells. For instance,the biphasic behavior of CSC marker expression with time may beattributed to the fraction of CSCs in the gel, which can depend on themicroenvironment, e.g., the stiffness of the surrounding environment andthe mesh size of the surrounding environment.

In one embodiment, the gel can be conjugated with one or more bindingpeptides that can affect the proliferation of CSCs, so as to elucidateadditional information about an encapsulated cell population and/or toselectively shut down/enhance the growth of CSCs in an encapsulated cellpopulation. For example, a CD44 binding peptide (e.g., RLVSYNGIIFFLK(SEQ ID NO.: 17) or a mutant thereof (VLFGFLKIYSRIN (SEQ ID NO.: 18))can be conjugated to the gel to inhibit proliferation of CSCs in thegel. A CD44 binding peptide (CD44BP) may be useful as CD44 expression isthe most widely used marker for characterization and identification ofbreast CSCs. CD44 is a cell membrane glycoprotein involved in cellmigration and adhesion. CD44 binds to many ECM ligands includinghyaluronic acid (HA), osteopontin, fibronectin and collagen. It alsobinds to matrix metalloproteinases (MMPs) and growth factors to promotetumor invasion and growth. As such, CD44 utilizes many signalingpathways to regulate cell behavior, and its activity depends onconformational changes and post-translational modifications after ligandbinding. CD44BP is a peptide derived from the D-domain of laminin o5chain. It binds to CD44 and inhibits lung colonization of tumor cells invivo but does not inhibit tumor cell proliferation when added to theculture medium.

CD44 has been used for CSC detection and targeting but the mechanism ofits involvement in the maintenance of CSCs is not clear. Antibodiesagainst CD44 are known to inhibit breast tumor growth and prevent cancerrecurrence. Anti-CD44 antibodies have been found to induce thedifferentiation of acute myeloid leukemia (AML) stem cells. In addition,a CD44 exon v6-specific antibody has been found to block the metastasisof rat pancreatic cancer cells. Therefore, utilization of CD44 bindingpeptide to regulate CSC population in the disclosed systems can providecritical information on the behavior of breast CSCs and/or on thebehavior of other cancer cells following depletion of the CSCs from acombined cell population.

Of course, the effect of other biochemical factors on cancer cells andin particular on CSCs can be examined in conjunction with the disclosedmatrices. For instance, binding peptides including an integrin bindingROD peptide (IBP) (e.g., GRGDS (SEQ ID NO.: 19) or a mutant thereofGRDGS (SEQ ID Na: 20)) or a fibronectin-derived heparin-binding peptide(FHBP) (WQPPRARI (SEQ ID NO.: 21) or a mutant thereof RPQIPWAR (SEQ IDNO.: 22)) can be conjugated to the gel to either enhance or deplete theCSC population in the gel and investigate in vitro the effect of abiochemical factor on encapsulated cells. These two peptides may beuseful as fibronectin is one of the major components of ECM thatmediates cell adhesion, and integrins are the major receptors on thecell surface that sense the environmental cues. In the specific examplesdescribed below, the results show that conjugation of FHBP to the gelmatrix enhanced tumorsphere formation by encapsulated 4T1 breast cancercells while CD44BP and IBP abolished sphere formation in vitro.

The RGD integrin binding peptide is present in many ECM components andcan be beneficially utilized to examine a cancer cell population, butthere are other binding motifs in the ECM that can alternatively beutilized. For example, fibronectin has RGD-independent heparin-bindingdomain in the C-terminus and binds to heparin sulfate proteoglycans onthe surface of tumor cells.

Moreover, biochemical factors that can be investigated by use of thesystem are not limited to binding proteins. The natural cellmicroenvironment is composed of many cellular and non-cellularcomponents such as cell binding proteins, growth factors, and nutrients,any of which can be incorporated in a matrix as disclosed herein.Moreover, variants of natural biochemical factors can be incorporated ina matrix including, without limitation, fragments, mutants, homologues,orthologues, analogues, etc. of naturally occurring proteins can beincorporated in a matrix, In addition, the biochemical factors to beexamined can be incorporated in the hydrogel matrix or can be includedin a cell culture medium in which the hydrogel and encapsulated cellpopulation can be incubated.

In one embodiment, the biochemical factor can include a cancer drug, Forinstance, a hydrogel matrix that encapsulates a cell population can beincubated in a cell culture medium that includes a cancer drug. Inaddition, the hydrogel matrix can include a conjugated peptide that canaffect the proliferation of cancer stem cells included in the cellpopulation (e.g., either enhance the proliferation of the CSCs ordeplete the population of the CSCs). Through study of the system, theusefulness of the biochemical factor as a cancer drug can be determined,

Any cancer drug and any type of cell population are encompassed herein,As utilized herein, the term “cancer drug” generally refers to any agentuseful to combat cancer. A non-limiting list of cancer drugs that caninvestigated by use of the disclosed matrices can be found in, forexample, U.S. Pat. No. 5,037,883, which is incorporated herein byreference. U.S. Pat, Nos. 6,348,209, 6,346,349, and 6,342,221 alsodescribe agents related to cancer drugs, all of which are incorporatedherein by reference.

Classes of cancer drugs encompassed herein include, but are not limitedto, chemotherapeutic agents, cytotoxins, antimetabolites, alkylatingagents, protein kinase inhibitors, anthracyclines, antibiotics,antimitotic agents (e.g. antitubulin agents), corticosteroids,radiopharmaceuticals, and proteins (e.g. cytokines, enzymes, orinterferons). Cancer drugs can include, for example, small moleculeorganic compounds, macromolecules, metal containing compounds, andcompounds or chelates that include radionuclides. In exampleembodiments, the cancer drug can be a small molecule organic compound.Specific examples include, but are not limited to docetaxel,gemcitabine, imatinib (Gleevecφ), 5-fluorouracil, 9-aminocamptothecin,amine-modified geldanamycin, doxorubicin, paclitaxel (Taxon, cisplatin,procarbazine, hydroxyurea, mesa e-chlorin, Gd(+3) compounds,asparaginase, and radionuclides (e.g I-131, Y-90, In-111, and Tc-99m).There are many cancer drugs known in the art and many continue to bedeveloped. In some embodiments, two or more cancer drugs can be examinedsimultaneously.

Due to the complex biochemical composition of the naturalmicroenvironment, it is difficult to study the role of individualfactors on cell behavior with in vitro models. By use of the disclosedinert system with controlled elastic modulus, any biochemical factor orcombination thereof can be investigated to determine the effect on thegrowth, proliferation and maintenance of cancer cells (for instance onthe sternness of breast CSCs) without the interference of other factors.Further, the ability of the system to selectively enrich CSCs in anencapsulated cell population can be of great benefit for drug testing.

Biochemical factors can be conjugated to the hydrogel matrix accordingto any suitable fashion that can maintain the activity of the factor.For instance, in one embodiment the biochemical factors can becovalently coupled to the macromer of the hydrogel matrix via reactivefunctionalization of the biochemical factor. Alternatively, abiochemical factor can be non-covalently coupled in the matrix, forinstance by charge-charge interaction or through encapsulation. Forinstance, a relatively large biochemical factor, e.g., a completeprotein, can be encapsulated in the matrix at the time of matrixformation and contained within the matrix as the mesh size of the matrixis smaller than the size of the biochemical factor.

The presently disclosed subject matter may be better understood withreference to the Examples set forth below.

EXAMPLE 1 Materials

Polyethylene glycol (PEG, nominal molecular weights 4.6 kDa),dichloromethane (DCM), N,N-dimethylformamide (DMF), diethyl ether, andhexane were purchased from Acros (Fairfield, Ohio). Calcium hydride,triethylamine (TEA), paraformaldehyde, 4,6-diamidino-2-phenylindole(DAPI), insulin, penicillin, and streptomycin were purchased fromSigma-Aldrich (St, Louis, Mo.). Basic fibroblast growth factor (bFGF)and epidermal growth factor (EGF) were purchased from Lonza (Allendale,N.J.). Bovine serum albumin (BSA) was obtained from JacksonlmmunoResearch (West Grove, Pa.). Dulbecco's phosphate-buffer saline(PBS), trypsin-EDTA, RPMI-160 cell culture medium, DMEM F12 medium,fetal bovine serum (FBS), Alexa Fluor@ 594 Phalloidin, and Quant-itPicoGreen dsDNA reagent kit were purchased from Invitrogen (Carlsbad,Calif.). Horse serum was purchased from PAA Laboratories (Etobicoke,Ontario) and DMEM-F12 was from Mediatech (Manassas, Va.). Spectro/Pordialysis tube (molecular weight cutoff 3.5 kDa) was purchased fromSpectrum Laboratories (Rancho Dominguez, Calif.), DCM was purified bydistillation over calcium hydride. All other solvents were reagent gradeand were used as received without further purification. Anti-CD44antibody (HCAM, DF1485) was from Santa Cruz Biotechnology (Santa Cruz,Calif.). Monoclonal Anti-BrdU antibody proliferation marker (produced inmouse) was obtained from Sigma-Aldrich. Fluorescent conjugated secondaryantibodies were obtained from Invitrogen. 4T1 mouse breast carcinoma andMCF7 human breast adenocarcinoma cell lines were received from theScripps Research Institute (La Jolla, Calif.) and American Type CultureCollection (ATCC, Manassas, Va.), respectively, The Live/Dead calcein AM(cAM) and ethidium homodimer-1 (EthD) cell viability/cytotoxicity kitwas purchased from Molecular Probes (Life Technologies, Grand Island,N.Y.).

Macromer Synthesis and Characterization

The PEG macromer was functionalized with acrylate groups to producepolyethylene glycol diacrylate (PEGDA) by the reaction of acryloylchloride with hydroxyl end-groups of PEG, as shown in FIG. 1A. TEA wasused as the reaction catalyst. Prior to the reaction, PEG was dried byazeotropic distillation from toluene to remove residual moisture. Thepolymer was dissolved in dried DCM in a reaction flask, the flask wasimmersed in an ice bath to cool the polymer solution and limit thetemperature rise from the exothermic reaction. In a typical reaction 5.6mL acryloyl chloride and 9.7 mL TEA, each dissolved in DCM, were addeddrop-wise to the reaction with stirring. The reaction was allowed toproceed for 12 h under nitrogen flow. After completion of the reaction,the solvent was removed by rotary evaporation and the residue wasdissolved in anhydrous ethyl acetate to precipitate the by-producttriethylamine hydrochloride salt. Next, ethyl acetate was removed byvacuum distillation; the macromer was re-dissolved in DCM andprecipitated twice in ice-cold ethyl ether. The macromer was dissolvedin dimethylsulfoxide (DMSO) and dialyzed against distilled deionized(DI) water to remove the by-products. The PEGDA product was freeze-driedand stored at -20° C. The chemical structure of the functionalizedmacromer was characterized by a Varian Mercury-300 1H-NMR (Varian, PaloAlto, Calif.) at ambient conditions with a resolution of 0.17 Hz, Thesample was dissolved in deuterated chloroform at a concentration of 5mg/mL and 1% v/v TMS was used as the internal standard. Hydrogelsynthesis and measurement of gel modulus

The PEGDA macromere were crosslinked in aqueous solution by UV initiatedradical polymerization with4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl) ketone (Irgacure 2959;CIBA, Tarrytown, N.Y.) photoinitiator. 5 mg of initiator was dissolvedin 1 mL PBS at 50° C. The macromer was dissolved in PBS by vortexing andheating to 50° C. To prepare 7.5, 10, 15, 20 and 25% PEGDA hydrogelprecursor solutions, 22.5, 30, 45, 60 and 75 mg of PEGDA macromer wasmixed with 278, 270, 255, 240 and 225 μL of the initiator solution,respectively, by vortexing for 5 min. For cell loading, 1.4×10⁵/mL 4T1cells suspended in PBS were added to the macromer solution and mixedgently with a glass rod. The hydrogel precursor solutions were degassedand transferred to a PTFE mold (5 cm x 3 cm x 750 μm), covered with atransparent glass plate, fastened with clips, and UV irradiated with aBLAK-RAY 100-W mercury long wavelength (365 nm) UV lamp (Model B100-AP;UVP, Upland, Calif.) for 10 min. Disc shape samples were cut from thegel using an 8 mm cork borer and swollen in PBS for 24 h at 37° C. Tomeasure the gel's elastic modulus, samples were loaded on the Peltierplate of the rheometer (TA Instruments, New Castle, DE) and subjected toa uniaxial compressive force at a displacement rate of 7.5 μm/s. Theslope of the linear fit to the stress-strain curve for 5-10% strain wastaken as the elastic modulus (E) of the gels.

Cancer Stem Cell Culture and Characterization

4T1 and MCF7 tumor cells were cultured in RMPI-1640 medium with 10% FBSunder 5% CO₂ at 37° C., Cells were trypsinized after reaching 70%confluency. PEGDA macromer was dissolved in PBS and sterilized byfiltration with a 0.2 μm filter. Next, 1.4 ×10⁵/mL 4T1 or MCF7 cellssuspended in PBS were added to the macromer solution with final PEGDAconcentrations ranging 5-25 wt %, and mixed gently with a pre-sterilizedglass rod. The cell-suspended hydrogel precursor solution wascrosslinked with UV for 10 min. After crosslinking, the gel was cut intodisks and incubated in stem cell culture medium in ultra-low attachmenttissue culture plates under 5% CO₂. The stem cell medium consisted ofDMEM-F12 supplemented with 0.4% bovine serum albumin (BSA), 5 μg/mLinsulin, 40 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL EGF,5% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

For growing tumorspheres in suspension, trypsinized 4T1 cells werecultured on ultra-low attachment tissue culture plates with stem cellculture medium under 5% CO₂ at 37° C. as described previously, The goldstandard for characterization of CSC tumorspheres for stemness is by theability to form tumor, To test for tumor formation, a stable 4T1 cellline that expressed luciferase (4T1-Luc) was established as known.Luciferase expression vector pGL4.50[Luc2/CMV/Hygro] (Prornega, Madison,Wis.) was transfected into 4T1 cells by Lipofectamine 2000 (Invitrogen),according to the manufacturer's instructions, to generate a cell lineexpressing luciferase as a reporter. After 24 h, cells were trypsinizedand cultured in RPMI-1640 medium with 400 μg/mL of hygromycin for 3weeks to generate 4T1-Luc cells. 4T1-Luc cells were cultured on adhesionplates with regular RPMI-1640 culture medium or on ultra-low attachmentplates with stern cell culture medium as describe above. After one week,cells were trypsinized and counted. Different number of cells (5000 and50,000 cells from adhesion plates or 500, 1,000 and 5,000 tumorspherecells from ultra-low attachment plates) was injected subcutaneously insyngeneic Balb/C mice (6 mice/group). One week after inoculation, 100 μLof D-Luciferin (30 mg/mL, Caliper, Hopkinton, Mass.) was injectedsubcutaneously and mice were imaged 10 min after Luciferin injection byCaliper's IVIS Spectrum imaging system (Caliper Life Sciences,Hopkinton, Mass.).

Cell Imaging and Determination of Cell Number

To determine cell viability, gels were stained with cAM/E hD live/deaddyes 2 days after encapsulation to image live and dead cells,respectively. Stained samples were imaged with an inverted fluorescentmicroscope (Nikon Eclipse Ti-ε, Nikon, Melville, N.Y.). Cell viabilitywas quantified by dividing the image into smaller squares and countingthe number of live and dead cells. At each time point, the gel sampleswere removed from the culture media and stained for imaging, Sampleswere rinsed twice with PBS and fixed with 4% paraformaldehyde for 3 h.After fixation, cells were permeabilized using PBS containing 0.1%Triton X-100 for 5 min. After rinsing, cells were incubated with Alexa488 phalloidin (1:200 dilution) and DAPI (1:5000 dilution) to stainactin filaments of the cell cytoskeleton and cell nuclei, respectively.Stained samples were imaged with a Nikon Eclipse Ti-ε invertedfluorescent microscope. For visualization of cell uniformity, a confocalfluorescent microscope (Zeiss LSM-510 META Axiovert, Carl Zeiss,Germany) was used to obtain 2D images (90 μm thick layers) of thestained gels in the direction of thickness. For determination of cellnumber, the gel samples were homogenized, cells were lysed, and aliquotswere used to measure the double stranded DNA (dsDNA) content using aQuant-it PicoGreen assay. Briefly, an aliquot (100 μL) of the workingsolution was added to 100 μL of the cell lysate and incubated for 4 minat ambient conditions. The fluorescence of the solution was measuredwith a plate reader (Synergy HT, Bio-Tek, Winooski, Vt.) at emission andexcitation wavelength of 485 and 528 m nm, respectively. Measuredfluorescent intensities were correlated to cell numbers using acalibration curve constructed with 4T1 or MCF7 cells of knownconcentration ranging from zero to 105cells/mL.

BrdU Retention Assay and Immunofluorescent Imaging

BrdU label-retention was used to identify mammary cancer stem cells.Nonconfluent 4T1 cells were incubated with 10 μM of BrdU for 10 days tolabel the DNA by incorporating BrdU into replicating DNA in place ofthymidine. Next, the BrdU labeled cells were encapsulated in the gel andincubated in stem cell culture medium to form tumorspheres as describedabove, At each time point, the retention of BrdU in the encapsulatedcells was imaged by immunofluorescent staining with Anti-BrdUantibodies. At each time point, tumorspheres encapsulated in the gelsamples were processed and stained for immunofluorescent imaging ofBrdU-labeled cells or CD44 marker. Gel Samples were fixed andpermeabilized for 3 h at 4° C. in PBS containing 4% paraformaldehyde and1% Triton X-100, followed by rinsing with PBS (3×10 min). Tumorspheroids were then dehydrated in an ascending series of methanol at 4°C. in PBS (25%, 50%, 75%, 95%, 30 min each and 100% for 5 h) andrehydrated in the same descending series and washed in PBS (3×10 min).Next, samples were blocked with PBS containing 0.1% Triton X-100 (PBST)and 3% BSA overnight at 4° C. and washed with PBS (2×15 min). Then,samples were incubated with primary antibodies (anti-CD44 antibody oranti-BrdU antibody) diluted in PBST on a gently rocking rotator at 4° C.overnight followed by rinsing with PBST (4×30 min). Samples were thenincubated with Alexa Fluor conjugated secondary antibodies for 2 h andrinsed with PBST (4×10 min). The cell nuclei were counterstained withDAPI (1:5000 dilution in PBS) and imaged with a Nikon Eclipse Ti-Einverted fluorescent microscope.

mRNA Analysis

Total cellular RNA of the gel samples was isolated using TRIzol(Invitrogen). 250 ng of the extracted purified RNA was reversetranscribed to cDNA by SuperScript II Reverse Transcriptase (Invitrogen)with the random primers. The obtained cDNA was subjected to real timequantitative polymerase chain reaction (RT-VCR) amplification withappropriate gene specific primers. RT-qPCR was performed to analyze thedifferential expression of CSC markers CD44 (4T1 and MCF7), CD24 (4T1),ABCG2 (4T1 and MCF7), and SCA1 (4T1) genes with SYBR green RealMasterMix(Eppendorf, Hamburg, Germany) using Bio-Rad iCycler PCR system (Bio-Rad,Hercules, Calif.). The expression level of GAPDH gene was used as aninternal control. The primers for real time PCR were designed by Primer3 software. The following forward and reverse primers synthesized byIntegrated DNA technologies (Coralville, Iowa) were used: mouse GAPDH:forward 5′-CAT GGC OTT CCG TGT TCC TA -3′ (SEQ ID NO: 1) and reverse5′-CCT GCT TCA CCA CCT TCT TGA-3′ (SEQ ID NO: 2); mouse CD44: forward5′-GAA TGT AAC CTG CCG CTA CG-3′ (SEQ ID NO: 3) and reverse 5′-GGA GGTGTT GGA COT GAC-3′ (SEQ ID NO: 4); mouse CD24: forward 5′-CTT CTG GCACTG CTC CTA CC-3′ (SEQ ID NO: 5) and reverse 5′-GAG AGA GAG CCA GGA GACCA-3′ (SEQ ID NO: 6); mouse ABCG2: forward 5′-AGC AGO AAG GAA AGA TCCAA-3′ (SEQ ID NO: 7) and reverse 5′-GGA ATA COG AGG CTG ATG AA-3′ (SEQID NO: 8); mouse SCA1: forward 5′-TGG ACA CTT CTC ACA CTA-3′ (SEQ ID NO:9) and reverse 5′-CAG AGC MG AGG GTC TOO AGO AG-3′ (SEQ ID NO: 10);human GAPDH forward 5′-GAG TCA ACG GAT TTG GTC GT-3′ (SEQ ID NO: 11) andreverse 5′-TTG ATT TTG GAG GGA TCT CG-3′ (SEQ ID NO: 12); human CD44forward 5′-GGC TTT CM TAG CAC OTT GC-3′ (SEQ ID NO: 13) and reverse5′-ACA CCC CTG TGT TGT TTG CT-3′ (SEQ ID NO: 14); human ABCG2 forward5′-CAC OTT ATT GGC CTC AGG AA-3′ (SEQ ID NO: 15) and reverse 5′-CCT GOTTGG AAG OCT CTA TG-3′ (SEQ ID NO: 16). The relative gene expressionlevels were quantified by the 2A--ddCT method as described. The relativegene expressions were expressed as fold difference compared with that attime zero.

Statistical Analysis

Data are expressed as means±standard deviation. All experiments weredone in triplicate. Significant differences between groups wereevaluated using a two-way ANOVA with replication test followed by atwo-tailed Student's t-test. A value of p<0.05 was consideredstatistically significant.

Macromer Characterization and Hydrogel Modulus

The reaction scheme for acrylation of PEG macromer and the NMR spectrumof PEGDA are shown in FIG. 1A and FIG. 1 B, respectively. The chemicalshifts with peak positions at 3.6 and 4.3 ppm were attributed to themethylene hydrogens (=CH₂) of PEG attached to ether (—CH₂——CH₂) andester (—CH₂—OOC—) groups, respectively, The shifts with peak positionsfrom 5.85 to 6.55 ppm (see inset in FIG. 1B) were attributed to thevinyl hydrogens (—CH═CH₂—) of the acrylate group at the end of eachmacromer arm as follows: Peak positions in the 5.82-5.87 ppm range wereassociated with the trans proton of unsubstituted carbon of the Ac;those in the 6.10-6.20 ppm range corresponded to the protons bonded tomonosubstituted carbon of the Ac; and those in the 6.40-6.46 ppm rangewere associated with the proton of unsubstituted carbon of the acrylategroup. The number of acrylate groups per rnacromer was determined fromthe ratio of NMR shifts between 5,85 and 6,55 ppm (acrylate hydrogens)to those at 3.6 and 4,2 ppm (PEG hydrogens).46, 47 Based on NMR results,the average degree of acrylation was 89% and there was on average 18±0.1acrylates in the PEGDA macromer. Disk shape hydrogels with 8 mm diameterand 750 μm thickness were fabricated for determination of elasticmodulus and cell encapsulation. The effect of macromer concentration andincubation time on the elastic modulus of PEGDA hydrogel, loaded with4T1 cells, is shown in FIG. 2. Macromer concentration in the precursorsolution affected the elastic modulus of the hydrogel. The elasticmodulus increased from 2.5±0,7 kPa to 5.3±1.4, 26.1±6.9, 47,5±11.6 and68.6±9.3 kPa as the macromer concentration was increased from 5% to 10,15, 20 and 25%, respectively. The elastic modulus of the gels, withencapsulated 4T1 cells, did not change significantly during 14 days ofincubation in the cell culture medium, as shown in FIG. 2.

Tumorsphere Characterization

Tumorsphere formation on ultra-low attachment plates is a commonly usedmethod to enrich CSCs in vitro, while the gold standard forcharacterization of CSC tumorspheres is by the ability to form tumor invivo, 4T1-Luc cells were cultured on regular adhesion plates orultra-low attachment plates. After one week, cells in monolayers(adhesion plates) and in spheres (ultra-low attachment plates) werecollected and subcutaneously inoculated in syngeneic Balblc mice. Tumorformation in mice was determined by imaging the expression of luciferaseone week after inoculation. FIG. 3 compares tumor formation by cells onadhesion plates (Panel A) and cells from tumorspheres on ultra-lowattachment plates (Panel B). The left and right images in Panel A arefor 5000 and 50,000 4T1-Luc cells on adhesion plates. The left, center,and right images in Pane! B are for 500, 1000, and 5000 4T1-Luc cellsfrom tumorspheres on ultra-low attachment plates. According to theimages in FIG. 3, 1000 tumorsphere cells were sufficient to form a tumorin vivo while it required 50,000 regular tumor cells to form a tumor.These results demonstrate that tumorspheres formed by 4T1 cells in vitrohad enriched CSC subpopulation.

Tumorsphere Formation in Hydrogel

4T1 tumor cells were encapsulated in PEGDA hydrogels with elastic moduliranging from 2 to 70 kPa and cultured in stem cell medium for 2 weeks.Images of live and dead cells 2 days after encapsulation in PEGDA gelswith moduli of 2.5, 5.3, 26.1 and 47.5 kPa are shown in FIG. 4A, FIG.4B, FIG. 4C, and FIG. 4D, respectively. Based on image analysis, thepercent viable cells for 2.5, 5.3, 26, 1, 47.5 and 68.6 kPa gels were94±4, 91±3, 92±3, 90±4, and 89±4, respectively. These results show thatthe gel modulus did not have a significant effect on viability of 4T1cells after encapsulation. To determine cell uniformity and viability, aconfocal microscope was used to image cells in the direction ofthickness and the results are shown in images FIG. 4E1 through FIG. 4E84. Images in FIG. 4E show uniform cell seeding and cell viability withinthe gel in the thickness direction.

Fluorescent images in FIG. 5 show the extent of cell aggregation andspheroid formation with incubation time for 4T1 gels with modulus of 2.5kPa (FIG. 5, 1^(st) column), 5.3 kPa (FIG. 5, 2^(nd) column), 47.5 kPa(FIG. 5, 3 ^(rd) column), and MCF7 gel with 5.3 kPa (FIG. 5, 4^(th)column). Rows 1, 2, 3, and 4 in FIG. 5 correspond to incubation times of5, 8, 11, and 14 days, respectively. Spheroid formation was observed insoft gels with moduli of 2.5 and 5.3 kPa as early as day 5 (1^(st),2^(nd), and 4^(th) columns in FIG. 5) while cells in the more stiff gelswith modulus of 47.5 kPa and higher remained as single cells or smallcell aggregates (<25 μm). MCF7 human breast cancer cells also formedspheres when encapsulated in the gel with modulus of 5.3 kPa (FIG. 5,4^(th) column). At any time point, size of the tumorspheres in the 5.3kPa gel was higher than that of 2.5 kPa gel. Lower magnification imagesin FIG. 6 show the number density of 4T1 and MCF7 tumorspheres in PEGDAgels with elastic moduli of 2.5 kPa, 5.3 kPa, 26.1 kPa, and 47.5 kPaafter 8 days of incubation. According to FIG. 6, the tumorsphere sizeand number density initially increased with increasing matrix modulusfrom 2.5 to 5.3 kPa and then decreased when modulus was increased to26.1 and 47.5 kPa. Tumorspheres with diameter >100 μm was observed onlyin the gels with modulus of 2.5 and 5.3 kPa but the fraction of largetumorspheres (>100 μm) was significantly higher in the 5.3 kPa gel. Itshould be noted that the size of MCF7 spheroids in the gels was lessthan that of 4T1.

Tumorsphere Size and Number Density

The effect of hydrogel modulus on average tumorsphere diameter and sizedistribution with incubation time is shown in FIG. 7A and FIG. 7B for4T1 cells and FIG. 7D and FIG. 7E for MCF7 cells, respectively. Theaverage 4T1 tumorsphere diameter increased from 10 μm at day zero to 80,140, 30, 15, and 10 μm after 14 days as the gel modulus increased from2.5 kPa to 5.3, 26.1, 47.5, and 68.6 kPa, respectively, whiletumorsphere diameter for MCF7 cells increased from 8 μm at day zero to60, 90, 29, 13, and 11 μm after 14 days. For 4T1 cells in the softestgel (2.5 kPa modulus), 25% of the cell aggregates at day 8 had <20 μmsize (single cell fraction) while there was no single cell subpopulationin the gel with 5.3 kPa modulus. For MCF7 cells after 8 days, 28% and 3%of the cell aggregates had <20 μm size in 2.5 and 5.3 kPa gels,respectively. Furthermore, for the gel with 5.3 kPa modulus, 23% and 8%of 4T1 tumorspheres and 8% and 3% of MCF7 tumorspheres had size in therange of 80.120 and 120-200 μm, respectively. The fraction oftumorspheres with 0-20 μm diameter (single cell fraction) increased withincreasing gel modulus for 4T1 and MCF7 cells and all of the cells inthe highest modulus gel (68.6 kPa) remained as single cells.

The number density of viable 4T1 and MCF7 cells in PEGDA gels withdifferent moduli is shown in FIG. 7C and FIG. 7F, respectively. The cellcount increased with time for all groups but the gels with moduli of 2.5and 5.3 kPa had the highest cell count at all time points. At each timepoint, the change in cell count with gel modulus was bimodal, that isthe cell count initially increased for moduli of 2.5 and 5.3 kPa andthen decreased for gels with moduli >26 kPa. At day 14, the 5.3 kPa gelhad 3 fold higher 4T1 cell and 1.3 fold higher MCF7 cell than the 2.5kPa gel; the 5.3 kPa gel had 10 fold higher 4T1 and MCF7 cells thanthose gels with >26 kPa modulus. In general, the gel modulus has similareffects on 4T1 and MCF7 cells. These results demonstrate that the gelwith modulus of 5.3 kPa had the highest potential for tumorsphereformation in the absence of ligand-receptor interactions.

Tumorsphere Marker Expression

One of the unique properties of CSCs is asymmetrical division andretention of DNA labeling. Based on this feature, BrdU retention is acommonly used method to characterize CSCs. 4T1 cells were labeled withBrdU before encapsulation in the PEGDA gel with 5.3 kPa modulus and theintensity of BrdU staining was compared with those cells cultured onultra-low attachment plates. FIG. 8 compares BrdU staining of 4T1 cellsin suspension culture on ultra-low attachment plates (FIG. 8A and FIG.80) with those encapsulated in PEGDA hydrogels (FIG. 8B and FIG. 8D).FIG. 8A an dFIG. 8B were obtained after 8 days of culture while FIG. 8Cand FIG. 8D were obtained after 14 days. After 8 and 14 days, cellsencapsulated in the gel displayed higher level of BrdU retention thanthose in suspension cultures, suggesting that the encapsulatedtumorspheres had higher fraction of CSCs.

The expressions of breast CSC markers for tumorspheres grown in PEGDAgels with different moduli are shown in FIG. 9. FIG. 9A through FIG. 9Dshow the expression of CD44, CD24, ABCG2, and SCA1 for 4T1 cells andFIG. 9E and FIG. 9F show the expression of CD44 and ABCG2 for MCF7cells. ABCG2 of ABC transporter proteins is responsible for CSC drugresistant and SCA1 (stem cell antigen-1) is a cell surface protein knownto be associated with breast CSCs.48-50 0044 and ABCG2 are well-studiedmarkers in both mouse and human breast cancer stem cells. Although CD24−is also a marker often used as a breast CSC marker, recent studiesindicate that both CD44/CD24− and CD44+/CD24+ cells display CSCphenotypes in MCF7 cells. SCA1 is a murine stem cell marker and it isunclear whether SCA1 is a CSC marker in human cancer cells. In addition,the coding sequence of human SCA1 is not well defined. Therefore, onlythe expression of CD44 and ABCG2 markers were examined for MCF7 cells.4T1 cells in the gel with elastic modulus of 5.3 kPa had the highest0044 expression and lowest 0024 expression for all time points. CD44expression of 4T1 and MCF7 cells initially increased and reached amaximum at day 8 for 2.5 and 5.3 kPa gels and at day 11 for 26.1 kPagel. CD44 expression then decreased with incubation time. 4T1 and MCF7cells encapsulated in the gels with moduli of 47.5 and 68.6 kPa did notshow an increase in CD44 expression in 14 days. This biphasic markerexpression with time was also observed for ABCG2 marker in 4T1 and MCF7cells. At day 8, CD44 and ABCG2 expression of 4T1 cells increased by 2.2and 1.8 folds, respectively, with increase in gel modulus from 2.5 to5.3 kPa; those for MCF7 cells increased by 1.2 and 1.7 folds withincrease in gel modulus from 25 to 5.3 kPa.

To determine whether tumorspheres in the hydrogel had a higher level ofstem cell population than those formed on ultra-low attachment plates,CD44 immunostaining of the 4T1 cells encapsulated in PEGDA gels (5.3 kPamodulus) was compared with those cultured on ultra-low attachment platesafter 5, 8, and 11 days of culture, and the results are shown in FIG.10. Tumorspheres grown in the gel and on ultra-low attachment plate bothhad high level of CD44 staining after 5 and 8 days of culture and theintensity of CD44 staining started to decrease after 8 days of culturefor both groups. This was consistent with the CD44 mRNA data (see FIG.9). However, tumorspheres grown in the gel had a more intense CD44staining than those on ultra-low attachment plates for longer incubationtimes of 11 and 14 days, suggesting that the 3D microenvironment and gelstiffness modulated the maintenance of sternness in CSCs.

The results also show that 4T1 cells form higher number of tumorsphereswhen encapsulated in the PEG hydrogel compared to suspension cultures onlow-adhesion plates. There are two possible explanations for thisobservation. One explanation is that the 3D hydrogel culture system moreclosely mimics the in vivo tissue environment than the suspensioncultures with respect to the survival of CSCs. Evidence supporting thisnotion is that cancer cells have fewer number of cancer stem cells in 2Dcultures than the in vivo. The elastic retractive force of the gelnetwork can promote viability and proliferation of CSCs by enhancing FGFsignaling and AKT activation. The other explanation is that theretractive force of the gel can induce the transformation ofdifferentiated bulk cancer cells into CSCs. The transformation betweenCSCs and differentiated cancer cells is not unidirectional. Recentstudies indicate that inducing epithelial to mesenchymal transition(EMT) is sufficient to transform a differentiated cancer cell into aCSC. In the process of EMT, tumor cells undergo cytoskeletalreorganization with subsequent changes in cell adhesion. At themolecular level, the key features of EMT include the altered expressionof cell membrane proteins such as E-cadherin and β-catenin, and cellpolarity. It is known that mechanical properties as well as biochemicalcomposition in the tumor microenvironment play profound roles in EMT. Itis possible that the hydrogel stiffness shifts the balance of EMT byregulating the conformation of cell membrane receptors and cellpolarity. One example of such mechanism is EGFR signaling which has beenassociated with breast cancer stem cell maintenance, Compression of thecell membrane by the gel retractive force can shrink the interstitialspace and increase the local ligand and receptor concentrations, thusincreasing the autocrine EGFR signaling.

EXAMPLE 2 Materials

Polyethylene glycol (PEG, nominal molecular weights 4.6 kDa),dichloromethane (DCM), N,N-dimethylformamide (DMF),diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP),trifluoroacetic acid (TFA), triisopropylsilane (TIPS), diethyl ether,and hexane were purchased from Acros (Fairfield, Ohio). The Rink AmideNovaGel™ resin, all Fmoc-protected amino acids, and hydroxybenzotriazole(HOBt) were purchased from Novabiochem (EMD Biosciences, San Diego,Calif.). Calcium hydride, triethylamine (TEA), paraformaldehyde,4,6-diamidino-2-phenylindole (DAPI), insulin, penicillin, andstreptomycin were purchased from Sigma-Aldrich (St. Louis, Mo.).Acetomethoxy derivative of calcein (cAM) and ethidium homodimer (EthD)were purchased from Molecular Probes (Life Technologies, Grand Island,N.Y.). Basic fibroblast growth factor (bFGF) and epidermal growth factor(EGF) were purchased from Lonza (Allendale, N.J.), Bovine serum albumin(BSA) was obtained from Jackson lmmunoResearch (West Grove, Pa.).Dulbecco's phosphate-buffer saline (PBS), trypsin-EDTA, RPMI-160 cellculture medium, fetal bovine serum (FBS), Alexa Fluor® 594 Phalloidin,and Quant-it PicoGreen dsDNA reagent kit were purchased from Invitrogen(Carlsbad, Calif.). Horse serum and DMEM-F12 medium were purchased fromPAA Laboratories (Etobicoke, Ontario) and MediaTech (Manassas, Va.),respectively. Spectro/Por dialysis tube (molecular weight cutoff 3.5kDa) was purchased from Spectrum Laboratories (Rancho Dominguez,Calif.). DCM was purified by distillation over calcium hydride. Allother solvents were reagent grade and were used as received withoutfurther purification. The anti-Actin, anti-VEGFa and anti-Vimentinantibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,Calif.). Fluorescent conjugated secondary antibodies were obtained fromInvitrogen. 4T1 mouse breast carcinoma cell line was developed by Dr,Suzanne Ostrand-Rosenberg group and available from ATCC (Manassas, Va.).The cell line was characterized and purified by Dr. Ralph A, Reisfeld atthe Scripps Research Institute (La Jolla, Calif.). 4T1 cells were adonation from Dr. Reisfeld under a Material Transfer Agreement. MCF7human breast adenocarcinoma cell line and MCF10a non-tumorigenicepithelial cell line were obtained from ATCC.

Macromer Synthesis

The PEG gel was formed as described above. A hydrolytically degradableversion of the PEGDA gel or poly(ethylene glycol-co-lactide) acrylatemacromer (dPEGDA or LPELA) was also formed. In a first step, linear(LPEL) poly(ethylene glycol-co-lactide) macromers were synthesized bymelt ring-opening polymerization of lactide with LPEG and SPEG,respectively, as polymerization initiators and TOC as the reactioncatalyst. LPEG and SPEC were dried by azeotropic distillation fromtoluene prior to the reaction. The LA and PEG were added to a three-neckreaction flask equipped with an overhead stirrer, The LA:PEG molar ratiowas varied from 0 to 20 to synthesize macromonomers with differentlactide segment lengths. The reaction flask was heated to 120° C. withan oil bath under steady flow of dry nitrogen to melt the reactants.Next, 1 ml of TOC was added and the reaction was allowed to continue for8 h at 135° C. After the reaction, the product was dissolved in DCM andprecipitated in ice cold methanol followed by ether and hexane tofractionate and remove the unreacted monomer and initiator. Thesynthesized LPEL and SPEL macromers were vacuum-dried to remove anyresidual solvent and stored at −20° C. In the next step, the terminalhydroxyl groups of LPEL and SPEL macrorners were reacted with acryloylchloride to produce LPELA and SPELA macromonomers, respectively. Priorto the reaction, macromers were dissolved in DCM and dried by azeotropicdistillation from toluene to remove residual moisture. After coolingunder steady flow of nitrogen, the macromer was dissolved in DCM and thereaction flask was immersed in an ice bath. Equimolar amounts ofacryloyl chloride and TEA were added drop-wise to the solution to limitthe temperature rise of the exothermic reaction. The reaction wasallowed to proceed for 12 h. After the reaction, solvent was removed byrotary evaporation, the residue was dissolved in ethyl acetate toprecipitate the by-product triethylamine hydrochloride salt. Next, ethylacetate was removed by vacuum distillation, the macromer wasre-dissolved in DCM and precipitated twice in ice cold ethyl ether. Thesynthesized macromonomer was dissolved in DMSO and purified by dialysisto remove any unreacted acrylic acid. The LPELA and SPELA products weredried in vacuum to remove residual solvent and stored at −40° C.

Peptide Synthesis and Characterization

CD44 binding peptide (CD44BP), integrin-binding ROD peptide (IBP), andfibronectin-derived heparin-binding peptide (FHBP) as well as theirmutants, were synthesized manually on Rink Amide resin in the solidphase using a known procedure. The sequences of these peptides and theirmutants are listed in the table below.

Peptide name Sequence Scrambled (mutant) CD44BP RLVSYNGIIFFLKVLFGFLKIYSRIN (SEQ ID NO: 17) (SEQ ID NO: 18) IBP GRGDS GRDGS(SEQ ID NO: 19) (SEQ ID NO: 20) FHBP WQPPRARI RPQIPWAR (SEQ ID NO: 21)(SEQ ID NO: 22)

Briefly, the Fmoc-protected amino acid (6 eq.), DIC (6.6 eq.), and HOBt(12 eq.) were added to 100 mg resin and swelled in DMF (3 mL). Next, 0.2mL of 0.05 M DMAP was added to the mixture and the coupling reaction wasallowed to proceed for 4-6 h at 30° C. with orbital shaking. The resinwas tested for the presence of unreacted amines using the Kaiserreagent. If the test was positive, the coupling reaction was repeated.Otherwise, the resin was treated with 20% piperidine in DMF (2×15 min)and the next Fmoc-protected amino acid was coupled using the sameprocedure. After coupling the last amino acid, the peptides werefunctionalized with an acrylamide group directly on the peptidyl resinby coupling acrylic acid to the N-terminal amine group under conditionsused for the amino acid coupling reaction. The acrylamide-terminatedpeptide was cleaved from the resin by treating with 95% TFA/2.5%TIPS/2.5% water and precipitated in cold ether. Theacrylamide-terminated (Ac) peptides were further purified by preparativeHPLC on a 250×10 mm, 10 μm Xterra Prep RP18 column (Waters, Milford,Mass.) with a flow rate of 2 mL/min using a gradient 5--95% MeCN in 0.1%aqueous TFA at detection wavelength of 214 nm. The HPLC fraction waslyophilized and the product was characterized with a Finnigan 4500Electro Spray Ionization (ESI) spectrometer (Thermo Electron, Waltham,Mass.).

Hydrogel Synthesis and Measurement of Modulus

The PEGDA macromer was crosslinked in aqueous solution to form a gel byultraviolet (UV) initiated radical polymerization with4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl) ketone (Irgacure 2959;CIBA, Tarrytown, N.Y.) photoinitiator. Five mg of initiator wasdissolved in 1 mL PBS at 50° C. The macromer was dissolved in PBS byvortexing and heating to 50° C. To prepare 10% PEGDA hydrogel precursorsolution, 30 mg PEGDA macromer was mixed with 270 mL of the initiatorsolution. The hydrogel precursor solution was degassed and transferredto a Teflon mold (5 cm×3 cm×500 μm), covered with a transparent glassplate and fastened with clips. Then, the assembly was irradiated with aBLAK-RAY 100-W mercury long wavelength (365 nm) UV lamp (Model B100-AP;UVP, Upland, Calif.) for 10 min. Next, disc shape samples were cut fromthe gel using an 8 mm cork borer and swollen in PBS for 24 h at 37° C.To measure the elastic modulus of the gel, samples were loaded on thePeltier plate of a rheometer (TA Instruments, New Castle, DE) andsubjected to uniaxial compressive force at a displacement rate of 7,5μm/s. The slope of the linear fit to the stress-strain curve for 5-10%strain was taken as the elastic modulus (E) of the gel.

Cancer Stem Cell Culture and Cell Encapsulation in the Hydrogel

The tumor cells were cultured in RMPI-1640 medium with 10% FBS under 5%CO₂ at 37° C. Cells were trypsinized after reaching 70% confluency. Thesynthesized acrylamide-terminated peptides were added to the PEGDAmacromer solution and the mixture was sterilized by filtration (220 nmfilter). Next, 1.4×10⁵/ml cells (4T1, MCF7, or MCF10a) were added to themacromer solution and mixed gently with a pre-sterilized glass rod. Thecell-suspended hydrogel precursor solution was crosslinked with UV for10 min as described above. After cross-linking, the gel was cut intodisks and incubated in stem cell culture medium on ultra-low attachmenttissue culture plates under 5% CO₂. The stem cell medium consisted ofDMEM-F12 supplemented with OA% BSA, 5 μg/ml insulin, 40 ng/ml bFGF, 20nglml EGF, 5% horse serum, 100 U/ml penicillin, and 100 μg/mIstreptomycin. For growing tumorspheres in suspension, trypsinized cells(4T1 or MCF7) were cultured on ultra-low attachment tissue cultureplates with stem cell culture medium under 5% CO₂ at 37° C.

Cell Imaging and Determination of Cell Number

To determine cell viability, gels were stained with cAM and EthD dyesafter cell encapsulation to image live and dead cells, respectively.Stained samples were imaged with an inverted fluorescent microscope(Nikon Eclipse Ti-ε, Nikon, Melville, N.Y.). Cell viability wasquantified by dividing the image into smaller squares and counting thenumber of live and dead cells manually. At each time point, three gelsamples were removed from the culture medium and stained for imaging.For imaging the encapsulated cells, gels were rinsed twice with PBS andfixed with 4% paraformaldehyde for 3 h. After fixation, cells werepermeabilized using PBS containing 0.1% Triton X-100 for 5 min. Afterrinsing, cells were incubated with Alexa 488 phalloidin (1:200 dilution)and DAPI (1:5000 dilution) to stain actin filaments of the cellcytoskeleton and cell nuclei, respectively. Stained samples were imagedwith a Nikon Eclipse Ti-ε inverted fluorescent microscope. Fordetermination of cell number, the gel samples were homogenized, cellswere lysed, and aliquots were used to measure the double stranded DNA(dsDNA) content using a Quant-it PicoGreen assay. Briefly, an aliquot(100 μL) of the working solution was added to 100 μl of the cell lysateand incubated for 4 min at ambient conditions. The fluorescence of thesolution was measured with a plate reader (Synergy HT, Bio-Tek,Winooski, Vt.) at emission and excitation wavelength of 485 and 528 nm,respectively. Measured fluorescent intensities were correlated to cellnumbers using a calibration curve constructed with cells of knownconcentration ranging from zero to 10⁵ cells/mL.

Real Time PCR Analysis

Total cellular RNA of the gel samples was isolated using TRIzol(Invitrogen). 250 ng of the extracted purified RNA was reversetranscribed to cDNA by SuperScript II Reverse Transcriptase (Invitrogen)with the random primers. The obtained cDNA was subjected to real timequantitative polymerase chain reaction (RT-qPCR) amplification with theappropriate gene specific primers. RT-qPCR was performed to analyze thedifferential expression of CSC markers CD44, CD24, ABCG2, and SCA1 geneswith SYBR green RealMasterMix (Eppendorf, Hamburg, Germany) usingBio-Rad iCycler PCR system (Bio-Rad, Hercules, Calif.). The expressionlevel of GAPDH gene was used as an internal control. The primers forreal time PCR were designed by Primer 3 software, The forward andreverse primer sequences, listed in the table below, were synthesized byIntegrated DNA technologies (Coralville, Iowa). The relative geneexpression levels were quantified by the 2̂(−ΔΔCT) method. Briefly, ACTwas calculated as ΔCT=CT^(target gene)−CT^(GAPDH). ΔΔCT of the targetgene was calculated as 66ΔCT=ΔCT^(experimental group)−ΔCT^(refererence group). The reference wasthe first time point (right after cells were encapsulated in the gel).The relative gene expression (fold-change compared to the reference timepoint) was calculated as 2̂(−ΔΔCT).

PCR Primer Forward Reverse mouse GAPDH 5′-CATGGCCTTCCGTGTTCC TA-3′5′-CCTGCTTCACCACCTTCTTGA-3′ (SEQ ID NO: 1) (SEQ ID NO: 2) mouse CD445′-GAA TGTAACCTG CCGCTACG-3′ 5′-GAGGTGTTGGACGTGAC-3′ (SEQ ID NO: 3)(SEQ ID NO: 4) mouse CD24 5′-CTTCTGGCACTGCTCCTACC-3′5′-GAGAGAGAGCCAGGAGACCA-3′ (SEQ ID NO: 5) (SEQ ID NO: 6) mouse ABCG25′-AGCAGCAAGGAAAGATCCAA-3′ 5′-GGAATACCGAGGCTGATGAA-3′ (SEQ ID NO: 7)(SEQ ID NO: 8) mouse SCA1 5′-TGGACACTTCTCACACTA-3′5′-CAGAGCAAGAGGGTCTGCAGGAG-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)mouse E-Cadherin 5′-ACTGTGAAGGGACGGTCAAC-3′ 5′-GGAGCAGCAGGATCAGAATC-3′(SEQ ID NO: 32) (SEQ ID NO: 23) mouse N-Cadherin5-GGGACAGGAACACTGCAAAT-3′ 5-CGGTTGATGGTCCAGTTTCT-3′ (SEQ ID NO: 24)(SEQ ID NO: 25) mouse integrin αV 5′-GCTTAAAGGCAGATGGCAAC-3′5′-AAATGGTGATGGGAGTGAGC-3′ (SEQ ID NO: 26) (SEQ ID NO: 27)mouse integrin β3 5′-TGACATCGAGCAGGAGGTGAAAAG-3′5′-GAGTAGCAAGGCCCAATGAGC-3′ (SEQ ID NO: 28) (SEQ ID NO: 29) mouse EGFR5′-CAGTGGGCAACCCTGAGTAT-3′ 5′-GGGCCCTTAAATATGCCATT-3′ (SEQ ID NO: 30)(SEQ ID NO: 31) human GAPDH 5′-GAGTCAACGGATTTG GTCGT-3′5′-TTGATTTTGGAGGGATCTCG-3′ (SEQ ID NO: 11) (SEQ ID NO: 12) human CD445′-GGCTTTCAATAGCACCTTGC-3′ 5′-ACACCCCTGTGTTGTTTGCT-3′ (SEQ ID NO: 13)(SEQ ID NO: 14) human ABCG2 5′-CACCTTATTGGCCTCAGGAA-3′5′-CCTGCTTGGAAGGCTCTATG-3′ (SEQ ID NO: 15) (SEQ ID NO: 16)

Flow Cytometry Analysis

Cells encapsulated in the gel were fixed with 4% paraformaldehyde for 30min followed by washing with PBS. Next, the gel was incubated inoxidative degradation solution (0,1M CoCI in 20% hydrogen peroxide).After the gel was degraded, cells were washed three times with cold PBScontaining 5% BSA. MCF7 cells were incubated with phycoerythrin (PE)mouse anti-human CD24 and fluorescein isothiocyanate (FITC) mouseanti-human CD44 (BD Biosciences, Franklin Lakes, N.J.), and 4T1 cellswere incubated with PE-anti-mouse CD24 and FITC-anti-mouse CD44(eBioscience, San Diego, Calif.) in 100 pi PBS with 5% BSA for 45 min onice in the dark. Cells were then washed with cold PBS with 5% BSA threetimes and analyzed by a flow cytometer (FC500, Beckman Coulter, Brea,Calif.). Flow cytometry was done multiple times on each sample toascertain reproducibility of the results.

Western Blot

The cell encapsulated gel was washed with PBS and homogenized in RIPAbuffer (1% NP4O, 1% SDS, 150mM NaCl, 20 mM Tris-Cl pH7.4, 1 mM EDTAprotease inhibitors) to extract the proteins. The homogenized sample wascentrifuged for 5 min to isolate total proteins. Next, proteins wereseparated by standard SDS-PAGE using Mini-gel system (Bio-Rad) andtransferred to a nitrocellulose membrane by the semi-dry transferapparatus (Bio-Rad). Membranes were incubated in the blocking buffer (5%fat-free dry milk in TBST buffer) at ambient conditions for 1 h followedby incubation with primary antibodies (1:200-1:2000) overnight at 4° C.After washing, the membrane was incubated with HRP-conjugated secondaryantibodies for 1 h at ambient conditions. After extensive washing withTBST, the membrane was incubated with ECL detection reagents and exposedto an X-ray film. The intensity of the band was quantified with thelmage-J software (National Institutes of Health, Bethesda, MD).

Tumor Growth in vivo and Measurement

To test tumor formation ability of 4T1 cells in the hydrogel, the cellencapsulated gels were cultured in vitro in the stem cell medium for 9days as described above. After tumorsphere formation, gel piecescontaining 1×10⁵ tumorsphere cells were implanted subcutaneously insyngeneic Balb/C mice (6 mice/group). Groups included 4T1 tumorspherecells, grown on ultra-low attachment plates, and injected subcutaneously(control group), degradable version of the PEGDA gel (dPEGDA) withouttumor cells (control group), 4T1 cells encapsulated in the dPEGDA andcultured in vitro for 9 days prior to implantation, and 4T1 cellsencapsulated in CD44BP-conjugated dPEGDA gel and cultured in vitro for 9days prior to implantation. When tumors became measurable, tumor sizeand growth rate were measured and calculated. Mice were euthanized whentumor volume reached above 1000 mm³ or 4 weeks after inoculation.

Statistical Analysis

Data were expressed as means±standard deviation. Significant differencesbetween groups were evaluated using a two-way ANOVA with replicationtest followed by a two-tailed Student's t-test. To account for multiplepair comparisons, p-values from the t-test were corrected using FalseDiscovery Rate (FOR) method. A value of p<0,05 was consideredstatistically significant.

Sphere Formation in PEGDA Hydrogel

Fluorescent images in FIG. 11A, FIG. 11B, and FIG. 11C show that 4T1 andMCF7 cancer cells encapsulated in the gel formed spheres but not thenormal MCF10a cells, suggesting that the spheres originated from the CSCsub-population of 4T1 and MCF7 cancer cells. The cell number density,sphere size, and size distribution for 4T1, MCF7 and MCF10a cellsencapsulated in the gel after 6 and 9 days incubation in stem cellculture medium are shown in FIG. 110 through FIG. 11 F. The cell densityof 4T1 and MCF7 cells significantly increased for both time points,while that of MCF10a remained at a low level (FIG. 110), suggesting thatthe PEGDA gel promoted the proliferation of tumor cells, but not thenormal cells. The density of 4T1 tumorspheres was slightly higher thanthat of MCF7 after 6 or 9 days of incubation. 4T1 cells also formedlarger spheres than MCF7 as shown in FIG. 11E. After 9 days of culturingin the gel, nearly 40% of the 4T1 spheres were larger than 80 μm whilemost of the MCF7 spheres were between 40 and 80 μm (FIG. 11F). MCF10aremained as single cells in the gel with size smaller than 20 μm.

The expressions of breast CSC markers CD44, CD24, and ABCG2 for theencapsulated cells are shown in FIG. 11G through FIG. 11I, respectively.After 6 days of incubation, CD44 expression level in 4T1 and MCF7 cellsincreased by 10 fold of the initial level (FIG. 11G). CD44 expressionwas further increased in 4T1 cells 9 days after encapsulation. However,as reported previously, the expression of CD44 in 4T1 and MCF7 cellsstarted to decrease after 11 days of incubation irrespective of theextent of cell viability and the increase in tumorsphere size. Theexpression of CD24 RNA was significantly reduced in 4T1 cells butincreased slightly in MCF7 (FIG. 11 H). Although CD44+/CD24− cells areconsidered breast CSCs, the expression of CD24 as a CSC marker in MCF7is not conclusive. Previous studies have indicated that CD44+/CD24− andCD44+/CD24+ cells both display CSC phenotypes in MCF7 cells. Thediscrepancy may be due to different primers used for real time PCRquantification, and antibodies used for cell sorting as well as themethods used for analysis. The expression of ABCG2, a subunit of ABCtransporter that is responsible for the drug resistance of CSCs was alsoincreased in 4T1 and MCF7 cells (FIG. 111). On the other hand, theexpression of these markers in MCF10a cells did not change.

FIG. 12A, FIG. 12B and FIG. 12C show representative images of live anddead 4T1 cells encapsulated in PEGDA gels with 5 kPa modulus after 2(FIG. 12A). 6 (FIG. 12B) and 12 (FIG. 12C) days, respectively. Theinsets are the corresponding figures in the 70 kPa gel. For 5 kPa gel,cell viability after 2, 6, and 12 days increased from 91±3% to 94±4% and97±2%, respectively. For the high modulus 70 kPa gel, no turnorsphereformed and cell viability decreased from 89±4% at day 2 to 84±3% and78±2% at days 6 and 12, respectively. Based on these results, the effectof peptide conjugation on tumorsphere formation was investigated with4T1 cells in the PEGDA gel with 5 kPa modulus and incubation time of 9days.

The CD44+/CD24− marker expression is widely used for identification ofbreast CSCs. Flow cytometry analysis of MCF7 cells isolated from the gelis shown in FIG. 13. The percentage of CD44+/CD24− cells beforeencapsulation in the gel was 2% (FIG. 13A) but it increased to 53% (FIG.13B) and 76% (FIG. 13D after 3 and 8 days incubation in the gel,respectively. However, the percent CD44+/CD24− cells decreased to 27%after 11 days incubation in the gel (FIG. 13D). These results areconsistent with previous results in which the CD44 mRNA expression of4T1 and MCF7 cells initially increased with time, then began to decreaseafter 14 days of incubation in the gel. The flow cytometry resultsdemonstrate that the percentage of CSCs in the population of cellsencapsulated in the gel increased dramatically after 8 days withincubation time and tumorsphere formation. Since the percentage of livecells in the gel increased with incubation time (see FIG. 12), thedecrease in the percentage of CSCs at day 11 (FIG. 13D) was presumablydue to the differentiation of CSCs.

Effect of CD44 Binding Peptide on Tumorsphere Formation in Hydrogels

Groups included the PEGDA gel without peptide conjugation (control,labeled as Ctrl in FIG. 14), the gel with CD44BP or scrambled CD44BP(FIG. 14, s-CD44BP) dissolved in the gel and in the culture medium tomaintain constant peptide concentration (labeled as Dis in FIG. 14), andthe gel with CD44BP or s-CD44BP conjugated to the gel (covalentattachment, labeled as Conj in FIG. 14). Fluorescent images FIG. 14Athrough FIG. 14D show the tumorspheres formed in conj CD44BP, conjs-CD44BP, dis CD44BP, and dis s-CD44BP, respectively. Tumorsphereformation was abolished when 4T1 cells were encapsulated in the CD44BPconjugated gel, indicating the importance of CD44 signaling in themaintenance of CSCs, The effect of CD44BP was consistent with previousreports. However, CD44BP dissolved in the gel (FIG. 14C and FIG. 14D)did not inhibit sphere formation. These results suggested that CD44BPdid not function as a soluble chemokine to inhibit CSC proliferation butfunctioned within the insoluble part of the ECM. The effect of ascrambled CD44BP (s-CD44BP) was also tested. Conjugated or dissolveds-CD44BP had no significant effect on tumorsphere formation, indicatingthat bioactivity was specific to CD44BP.

FIG. 14E and FIG. 14F show the effect of CD44BP on cell number densityand sphere size of 4T1 cells encapsulated in the gel after 9 days ofincubation. The 4T1 cell density in the gel reached 14×10⁶/ml after 9days with 1.4×10⁵/ML initial cell seeding in the gel. The density of 471cells in the gel with CD44BP (conjugated or dissolved and with orwithout mutation) were lower compared with the gel without any peptide.However, cells in the conj CD44BP gel had the strongest effect on celldensity and completely abolished sphere formation (FIG. 14A). Theexpression of CSC markers, CD44, CD24, ABCG2 and SCA1 was alsodetermined and the results are shown in FIG. 14G through FIG. 14J,respectively. 4T1 cells in the CD44BP gel that formed spheres (conjs-CD44BP, dis CD44BP and dis s-CD44BP) had high expressions of CD44,ABCG2 and SCA1 and low expression of CD24. On the other hand, cells inthe conj CD44BP gel, which did not form tumorspheres, had decreasedexpressions of CD44. ABCG2 and SCA1, and increased expression of CD24.These results indicated that tumorsphere formation by 4T1 cells in thegel correlated with the CSC population.

Effect of CD44 Binding Peptide on Tumor Formation in viva

It is well established that tumor growth in vivo requires a permissiveenvironment that can support vascularization and matrix remodeling.Therefore, a degradable version of PEGDA gel (dPEGDA) was used toinvestigate the effect of CD44BP conjugated to the gel on tumorformation in vivo by the encapsulated 4T1 cells. Groups included 4T1tumorspheres injected directly without the gel, gels without cell, gelswithout peptide conjugation but with 4T1 tumorspheres, and gels with 4T1cells conjugated with CD44BP. The gels without cell did not from avisible tumor after 4 weeks (FIG. 15), Tumors became measurable after 10days with direct subcutaneous injection of 4T1 tumorspheres. 4T1tumorspheres in the gel without CD44BP conjugation also formed a tumorafter 13 days of inoculation. Even though the formation of tumor wasdelayed when cells were encapsulated in the gel, the growth rate (theslop of the tumor size curve) did not differ significantly between thegroup with 4T1 in PBS and 4T1 in the gel (FIG. 15). The observed lagtime in tumor formation for the encapsulated cancer cells is presumablyrelated to the degradation time of the gel and connection of the tumorcells to the surrounding tissue. However, 4T1 cells encapsulated in theconj CD44BP gel did not form a visible tumor after 4 weeks ofinoculation, indicating that CD44BP conjugated to the gel inhibitedtumor formation in viva

Comparing the Effect of CD44 Binding Peptide on Tumorsphere Formationwith Integrin and Heparin Binding Peptides

The effect of CD44BP on tumorsphere formation in the gel promptedtesting of other cell-binding peptides. IBP, an integrin receptorbinding peptide and FHBP, a heparin-binding domain of fibronectin thatbinds to cell surface heparin sulfate proteoglycans, were conjugated tothe gel. Groups included 4T1 cell seeded gel without peptideconjugation, the cell-seeded gel with CD44BP conjugation, thecell-seeded gel with IBP conjugation, and the cell-seeded gel with FHBPconjugation. For determination of marker expression, gels conjugatedwith a scrambled sequence of the peptides were also tested. Fluorescentimages in FIG. 16A through FIG. 16D show sphere formation by 4T1 cellsin the gels without peptide, with conj CD44BP, conj IBP, and conj FHBP,respectively. The IBP conjugation, similar to CD44BP, abolished 4T1tumorsphere formation in the gel (FIG. 16C). However, tumorsphereformation increased when 4T1 cells were encapsulated in the FHBPconjugated gel (FIG. 16D). Further characterization of the cells inthese gels (FIG. 16E through FIG. 16G) showed that the cells in IBP andCD44BP conjugated gels had reduced cell number, did not form sphere, andremained as single cells or small cell aggregates (<25 μm). On the otherhand, the cells in FHBP conjugated gel had higher cell number and largerspheres compared with those in the gels without peptide conjugation.

To determine whether the size and number density of tumorspheres in thegel correlated with the CSC sub-population, CD44 and CD24 expression ofthe cells in the peptide-conjugated gels were measured, 4T1 cells in thegels without any peptide conjugation and with FHBP conjugation hadelevated expression of CD44 marker while the cells in gels conjugatedwith CD44BP or IBP had decreased CD44 expression (see FIG. 16H). Moreimportantly, the CD44 expression in the cells encapsulated in FHBPconjugated gel was significantly higher than that without peptideconjugation. The expression of CD24 in those gels had an oppositepattern to that of CD44 (see FIG. 16H). In breast cancer, the expressionof epidermal growth factor receptor (EGFR) is also closely related tothe maintenance of CSCs. The expression of EGFR marker by 4T1 cellsencapsulated in the peptide-conjugated gels is shown in FIG. 16J.Similar to CD44 marker, the expression of EGFR was increased in thecells encapsulated in FHBP conjugated gel but decreased in CD44BP andIBP conjugated gels. Furthermore, conjugation of a mutant sequence ofthe peptides to the gel had insignificant or limited effect ontumorsphere formation and the expression of CSC markers, compared to thewild type (FIG. 16H through FIG. 16J).

The effect of cell binding peptides on CSC sub-population was furtherexamined in 4T1 cells by flow cytometry. The percentage of CD44+/CD24−cells in 4T1 cells cultured without gel encapsulation was about 6% (FIG.17A). This percentage doubled to 12% for cells encapsulated in the PEGDAgel without peptide conjugation (FIG. 17B). When 4T1 cells wereencapsulated in the gel conjugated with FHBP, the sub-population ofCD44+/CD24− cells was further increased to about 21% (FIG. 17C).Conversely, the fraction of CSC sub-population in the gel decreased tothe original level (5.4% for IBP and 6.5% for CD44BP) when 4T1 cellswere encapsulated in the gels conjugated with IBP (FIG. 17D) or CD44BP(FIG. 17E) that inhibited sphere formation. These results suggested thattumorsphere formation by 4T1 cells in the gel was related to the CSCsub-population.

Effects of Cell Binding Peptides on the Expression of other CSC RelatedMarkers

One of the pathways to transform differentiated cancer cells into CSCsis epithelial to mesenchymal transition (EMT). The hallmark of EMT isthe decreased expression of E-Cadherin and increased expression ofN-Cadherin. The expressions of E-Cadherin and N-Cadherin 3 and 9 daysafter cells were encapsulated in the peptide-conjugated gels are shownin FIG. 18A and FIG. 18B, respectively. At the early time point (3days), the expression of E-Cadherin was decreased while the expressionof N-Cadherin was increased in the gel with FHBP, suggesting that EMTwas a possible mechanism for the enhanced tumorsphere formation in thegel. However, at the later time point (9 days), the expression ofE-Cadherin in the cells grown in FHBP gel was much higher than that inother groups. This was probably due to sphere formation in the FHBP gel.E-Cadherin is a cell adhesion protein and its expression increases withincreased cell-cell interaction. Consistent with that, cells in the IBPand CD44BP gels, which did not form spheres, had low expressions ofE-Cadherin (FIG. 18B).

The importance of integrins in cancer and CSC maintenance is well known.Since RGD is an integrin binding peptide, we examined the expression ofintegrin αV and β[3, two integrin subunits required for RGD binding, andthe expressions are shown in FIG. 18C and FIG. 18D, respectively. Theexpression of integrin αV and β3 was reduced in cells grown in FHBP andCD44BP gels, even though the cells in FHBP gel formed spheres whilethose in CD44BP gel did not, Interestingly, the expression of integrinαV and β3 was significantly increased in the cells in IBP gel. It ispossible that blocking integrin signaling by RGD activates a feedbackloop to induce integrin expression. These results suggest that theexpression of integrin does not correlate directly with tumorsphereformation or CSC maintenance of 4T1 cells, Conjugation of a mutantsequence of the peptides to the gel had limited effect on the expressionof CSC markers, indicating that the effect was specific to the wildtype.

The expression of vimentin and VEGF, two other markers related toinvasive breast cancer and CSC maintenance, was also determined at theprotein level, as shown in FIG. 18E and FIG. 18F. The expression ofvimentin and VEGF was high in cells that formed spheres, and theirexpression correlated with the sphere size and number (FIG. 16E, FIG.16F),

In this study, we found that CD44BP inhibits tumorsphere formation onlywhen conjugated (covalently attached) to the gel. Dissolving the peptidein the gel or in the medium did not have an effect on tumorsphereformation. This result suggests that CD44BP does not act as a solublechemokine to block or activate CD44 signaling. We speculate that CD44BPinduces a conformational change in CD44 receptor through a mechanismlike receptor clustering.

Using the 3D PEGDA matrix with a certain stiffness, it has beendemonstrated that the cell adhesion CD44 binding peptide (CD44BP), RGDintegrin binding peptide (IBP), and fibronectin-derived heparin bindingpeptide (FHBP) can be individually conjugated to the inert PEGDA gel andtheir effect on the maintenance of breast cancer stern cells can beinvestigated without the interference of other factors. The CD44BP andIBP conjugated to the inert gel completely abolished tumorsphereformation by the encapsulated 4T1 breast cancer cells while FHBPenhanced tumorsphere formation compared to those without peptide. Theinert 3D hydrogel cell culture system provides a novel tool toinvestigate the individual effect of factors in the microenvironment onmaintenance of CSCs without the interference of other factors, Morespecifically, the 3D hydrogel cell culture system can be used toselectively enrich the cancer cells with CSC sub-population for instancefor the purpose of testing toxicity of chemotherapy agents against theCSC sub-population,

It will be appreciated that the foregoing examples, given for purposesof illustration, are not to be construed as limiting the scope of thisdisclosure. Although only a few exemplary embodiments have beendescribed in detail above, those skilled in the art will readilyappreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this disclosure. Accordingly, all such modifications areintended to be included within the scope of this disclosure which isdefined in the following claims and all equivalents thereto. Further, itis identified that many embodiments may be conceived that do not achieveall of the advantages of some embodiments, yet the absence of aparticular advantage shall not be construed to necessarily mean thatsuch an embodiment is outside the scope of the present invention.

What is claimed is:
 1. A method of forming a three dimensional hydrogelmatrix for supporting a cancer cell, the method comprising: combining aninert synthetic polymer with a crosslinking agent to form a precursorsolution; crosslinking the inert synthetic polymer via the crosslinkingagent to form the three dimensional hydrogel matrix, wherein theconcentration of the crosslinking agent and/or the concentration of theinert synthetic polymer is predetermined in the precursor solution suchthat the three dimensional hydrogel matrix has a predetermined elasticmodulus; and conjugating a peptide to the matrix, the peptide affectingthe growth, development, and/or proliferation of a cancer stem cell. 2.The method of claim 1, wherein the inert synthetic polymer comprisespolyethylene glycol, polyhydroxyethyl methacrylate,polyvinylolypyrrolidone, or polyvinyl alcohol.
 3. The method of claim 1,wherein the inert synthetic polymer is combined with and reacted withthe crosslinking agent prior to crosslinking the polymer.
 4. The methodof claim 1, further comprising encapsulating a population of cells inthe three dimensional hydrogel matrix, the population of cellscomprising cancer stem cells.
 5. The method of claim 4, the populationof cells further comprising cancer cells.
 6. The method of claim 5,wherein the cancer cells are breast cancer cells,
 7. The method of claim4, the population of cells further comprising tumor stroma cells.
 8. Themethod of claim 1, further comprising combining the polymer and thecrosslinking agent with an initiator.
 9. The method of claim 6, furthercomprising subjecting the polymer to radiation to initiate thecrosslinking.
 10. The method according to claim 1, wherein the precursorsolution includes the inert synthetic polymer in a concentration ofabout 10% by weight of the precursor solution or less and thepredetermined elastic modulus is about 10 kilopascals or less.
 11. Themethod according to claim 1, wherein the precursor solution includes theinert synthetic polymer in a concentration of from about 10% by weightof the precursor solution to about 20% by weight of the precursorsolution and the predetermined elastic modulus is from about 10kilopascals to about 30 kilopascals.
 12. The method according to claim1, wherein the precursor solution includes the inert synthetic polymerin a concentration of about 20% by weight of the precursor solution orgreater and the predetermined elastic modulus is about 30 kilopascals orgreater.
 13. The method of claim 1, wherein the peptide is a CD44binding peptide or a mutant thereof, an integrin binding peptide or amutant thereof, or a heparain binding peptide or a mutant thereof. 14.The method of claim 11, wherein the peptide is RLVSYNGIIFFLK (SEQ IDNO.: 17), VLFGFLKIYSRIN (SEQ ID NO.: 18), GRGDS (SEQ ID NO.: 19), GRDGS(SEQ ID NO.: 20), WQPPRARI (SEQ ID NO.: 21), or RPQIPWAR (SEQ ID NO.:22).
 15. The method of claim 1, further comprising incorporating abiochemical factor in the hydrogel matrix.
 16. A three dimensionalhydrogel matrix comprising a crosslinked inert synthetic polymer and apeptide conjugated to the matrix, the peptide affecting the growth,development, and/or proliferation of a cancer stern cell, the threedimensional hydrogel matrix having a predetermined elastic modulus. 17.The three dimensional hydrogel matrix of claim 16, wherein the threedimensional hydrogel matrix has an elastic modulus of from 2.5kilopascals to 70 kilopascals.
 18. The three dimensional hydrogel matrixof claim 16, wherein the three dimensional hydrogel matrix has anelastic modulus of from about 2.5 kilopascals to about 10 kilopascals.19. The three dimensional hydrogel matrix of claim 16, wherein thepeptide is a CD44 binding peptide or a mutant thereof, an integrinbinding peptide or a mutant thereof, or a heparin binding peptide or amutant thereof.
 20. The three dimensional hydrogel matrix of claim 19,wherein the peptide is RLVSYNGIIFFLK (SEQ ID NO.: 17), VLFGFLKIYSRIN(SEQ ID NO.: 18), GRGDS (SEQ ID NO.: 19), GRDGS (SEQ ID NO.: 20),WQPPRARI (SEQ ID NO.: 21), or RPQIPWAR (SEQ ID NO.: 22).
 21. The threedimensional hydrogel matrix of claim 16 further comprising a biochemicalfactor incorporated in the three dimensional hydrogel matrix.
 22. Thethree dimensional hydrogel matrix of claim 16, further comprising apopulation of cells encapsulated in the matrix, the population of cellscomprising cancer stem cells.
 23. The three dimensional hydrogel matrixof claim 22, the population of cells futher comrprising cancer cells.24. The three dimensional hydrogel matrix of claim 22, the population ofcells further comprising tumor stroma cells.
 25. A method for studying acell population, the method comprising: encapsulating a cell populationin a three dimensional hydrogel matrix, the three dimensional hydrogelmatrix having a predetermined elastic modulus, the hydrogel matrixincluding a peptide conjugated to the matrix, the peptide affecting thegrowth, development and/or proliferation of a cancer stem cell, the cellpopulation comprising cancer stem cells; and incubating the hydrogelmatarix encapsulating the cell population in a cell culture medium, thecell culture medium comprising a biochemical factor,
 26. The method ofclaim 25, wherein the biochemical factor is a cancer drug.
 27. Themethod of claim 25, wherein the peptide is a CD44 binding peptide or amutant thereof, an integrin binding peptide or a mutant thereof, or aheparin binding peptide or a mutant thereof.
 28. The method of claim 27,wherein the peptide is RLVSYNGIIFFLK (SEQ ID NO.: 17), VLFGFLKIYSRIN(SEQ ID NO,: 18), GRGDS (SEQ ID NO.: 19), GRDGS (SEQ ID NO.: 20),WQPPRARI (SEQ ID NO.: 21), or RPQIPWAR (SEQ ID NO.: 22),
 29. The methodof claim 25, the cell population comprising cancer cells.
 30. The methodof claim 25, the cell population comprising tumor stroma cells.